Commensal Bacteroidetes protect against Klebsiella pneumoniae colonization and transmission through IL-36 signalling

Abstract

The microbiota primes immune defences but the identity of specific commensal microorganisms that protect against infection is unclear. Conversely, how pathogens compete with the microbiota to establish their host niche is also poorly understood. In the present study, we investigate the antagonism between the microbiota and Klebsiella pneumoniae during colonization and transmission. We discover that maturation of the microbiota drives the development of distinct immune defence programmes in the upper airways and intestine to limit K. pneumoniae colonization within these niches. Immune protection in the intestine depends on the development of Bacteroidetes, interleukin (IL)-36 signalling and macrophages. This effect of Bacteroidetes requires the polysaccharide utilization locus of their conserved commensal colonization factor. Conversely, in the upper airways, Proteobacteria prime immunity through IL-17A, but K. pneumoniae overcomes these defences through encapsulation to effectively colonize this site. Ultimately, we find that host-to-host spread of K. pneumoniae occurs principally from its intestinal reservoir, and that commensal-colonization-factor-producing Bacteroidetes are sufficient to prevent transmission between hosts through IL-36. Thus, our study provides mechanistic insight into when, where and how commensal Bacteroidetes protect against K. pneumoniae colonization and contagion, providing insight into how these protective microorganisms could be harnessed to confer population-level protection against K. pneumoniae infection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The adult microbiota protects against colonization by antibiotic-resistant K. pneumoniae in the intestine but not the upper airways.
Fig. 2: Intestinal Bacteroidetes protect against K. pneumoniae colonization.
Fig. 3: Bacteroidetes protect against K. pneumoniae colonization in the intestine through IL-36 signalling and macrophages.
Fig. 4: Bacteroidetes require their CCFs to protect against K. pneumoniae colonization in the intestine.
Fig. 5: Proteobacteria primes upper airway defences through IL-17A, but encapsulation allows K. pneumoniae to overcome these defences.

Data availability

Microbiome sequencing data have been deposited at the National Center for Biotechnology Information, Sequence Read Archive (BioProject PRJNA579139). The data supporting the findings of the study are available in this article (see Source Data) and its Supplementary Information files, or from the corresponding author upon request.

References

  1. 1.

    Baumler, A. J. & Sperandio, V. Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535, 85–93 (2016).

  2. 2.

    Brown, R. L. & Clarke, T. B. The regulation of host defences to infection by the microbiota. Immunology 150, 1–6 (2017).

  3. 3.

    Honda, K. & Littman, D. R. The microbiome in infectious disease and inflammation. Annu. Rev. Immunol. 30, 759–795 (2012).

  4. 4.

    Clarke, T. B. Microbial programming of systemic innate immunity and resistance to infection. PLoS Pathog. 10, e1004506 (2014).

  5. 5.

    Keith, J. W. & Pamer, E. G. Enlisting commensal microbes to resist antibiotic-resistant pathogens. J. Exp. Med. 216, 10–19 (2019).

  6. 6.

    Gupta, N., Limbago, B. M., Patel, J. B. & Kallen, A. J. Carbapenem-resistant Enterobacteriaceae: epidemiology and prevention. Clin. Infect. Dis. 53, 60–67 (2011).

  7. 7.

    Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! an update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009).

  8. 8.

    Munoz-Price, L. S. et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Dis. 13, 785–796 (2013).

  9. 9.

    Nordmann, P., Naas, T. & Poirel, L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 17, 1791–1798 (2011).

  10. 10.

    Martin, R. M. & Bachman, M. A. Colonization, infection, and the accessory genome of Klebsiella pneumoniae. Front. Cell Infect. Microbiol. 8, 4 (2018).

  11. 11.

    Montgomerie, J. Z. Epidemiology of Klebsiella and hospital-associated infections. Rev. Infect. Dis. 1, 736–753 (1979).

  12. 12.

    Paczosa, M. K. & Mecsas, J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiol. Mol. Biol. Rev. 80, 629–661 (2016).

  13. 13.

    Podschun, R. & Ullmann, U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589–603 (1998).

  14. 14.

    World Health Organization. Antimicrobial resistance: global report on surveillance (World Health Organization, 2014).

  15. 15.

    Bagley, S. T. Habitat association of Klebsiella species. Infect. Control 6, 52–58 (1985).

  16. 16.

    Donskey, C. J. The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens. Clin. Infect. Dis. 39, 219–226 (2004).

  17. 17.

    Martin, R. M. et al. Molecular epidemiology of colonizing and infecting isolates of Klebsiella pneumoniae. mSphere 1, e00261–16 (2016).

  18. 18.

    The Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214 (2012).

  19. 19.

    Rose, H. D. & Schreier, J. The effect of hospitalization and antibiotic therapy on the Gram-negative fecal flora. Am. J. Med. Sci. 255, 228–236 (1968).

  20. 20.

    Bilinski, J. et al. Fecal microbiota transplantation in patients with blood disorders inhibits gut colonization with antibiotic-resistant bacteria: results of a prospective, single-center study. Clin. Infect. Dis. 65, 364–370 (2017).

  21. 21.

    Caballero, S. et al. Distinct but spatially overlapping intestinal niches for vancomycin-resistant Enterococcus faecium and carbapenem-resistant Klebsiella pneumoniae. PLoS Pathog. 11, e1005132 (2015).

  22. 22.

    Singh, R. et al. Fecal microbiota transplantation against intestinal colonization by extended spectrum beta-lactamase producing Enterobacteriaceae: a proof of principle study. BMC Res. Notes 11, 190 (2018).

  23. 23.

    Sorbara, M. T. et al. Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification. J. Exp. Med. 216, 84–98 (2019).

  24. 24.

    Lau, H. Y., Huffnagle, G. B. & Moore, T. A. Host and microbiota factors that control Klebsiella pneumoniae mucosal colonization in mice. Microbes Infect. 10, 1283–1290 (2008).

  25. 25.

    Madueno, A. et al. Risk factors associated with carbapenemase-producing Klebsiella pneumoniae fecal carriage: a case-control study in a Spanish tertiary care hospital. Am. J. Infect. Control 45, 77–79 (2017).

  26. 26.

    Mills, J. P., Talati, N. J., Alby, K. & Han, J. H. The epidemiology of carbapenem-resistant Klebsiella pneumoniae colonization and infection among long-term acute care hospital residents. Infect. Control Hosp. Epidemiol. 37, 55–60 (2016).

  27. 27.

    Bhargava, A. et al. Risk factors for colonization due to carbapenem-resistant Enterobacteriaceae among patients exposed to long-term acute care and acute care facilities. Infect. Control Hosp. Epidemiol. 35, 398–405 (2014).

  28. 28.

    Nelson, A. L., Barasch, J. M., Bunte, R. M. & Weiser, J. N. Bacterial colonization of nasal mucosa induces expression of siderocalin, an iron-sequestering component of innate immunity. Cell Microbiol. 7, 1404–1417 (2005).

  29. 29.

    Brown, R. L., Sequeira, R. P. & Clarke, T. B. The microbiota protects against respiratory infection via GM-CSF signaling. Nat. Commun. 8, 1512 (2017).

  30. 30.

    Atarashi, K. et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Cell 163, 367–380 (2015).

  31. 31.

    Donaldson, G. P. et al. Gut microbiota utilize immunoglobulin a for mucosal colonization. Science 360, 795–800 (2018).

  32. 32.

    Lee, S. M. et al. Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501, 426–429 (2013).

  33. 33.

    Rendueles, O., Garcia-Garcera, M., Neron, B., Touchon, M. & Rocha, E. P. C. Abundance and co-occurrence of extracellular capsules increase environmental breadth: implications for the emergence of pathogens. PLoS Pathog. 13, e1006525 (2017).

  34. 34.

    Clements, A. et al. Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides. J. Biol. Chem. 282, 15569–15577 (2007).

  35. 35.

    de Steenhuijsen Piters, W. A. et al. Dysbiosis of upper respiratory tract microbiota in elderly pneumonia patients. ISME J. 10, 97–108 (2016).

  36. 36.

    Yi, H., Yong, D., Lee, K., Cho, Y. J. & Chun, J. Profiling bacterial community in upper respiratory tracts. BMC Infect. Dis. 14, 583 (2014).

  37. 37.

    Kaspar, U. et al. The culturome of the human nose habitats reveals individual bacterial fingerprint patterns. Environ. Microbiol. 18, 2130–2142 (2016).

  38. 38.

    Pettigrew, M. M. et al. Upper respiratory tract microbial communities, acute otitis media pathogens, and antibiotic use in healthy and sick children. Appl. Environ. Microbiol. 78, 6262–6270 (2012).

  39. 39.

    Bassis, C. M. et al. Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals. mBio 6, e00037 (2015).

  40. 40.

    de Steenhuijsen Piters, W. A., Sanders, E. A. & Bogaert, D. The role of the local microbial ecosystem in respiratory health and disease. Philos. Trans. R Soc. Lond. B Biol. Sci. 370, 20140294 (2015).

  41. 41.

    Zhou, Y. et al. Biogeography of the ecosystems of the healthy human body. Genome Biol. 14, R1 (2013).

  42. 42.

    Harris, A. D. et al. Patient-to-patient transmission is important in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae acquisition. Clin. Infect. Dis. 45, 1347–1350 (2007).

  43. 43.

    Snitkin, E. S. et al. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci. Transl. Med. 4, 148ra116 (2012).

  44. 44.

    Ebino, K. Y. Studies on coprophagy in experimental-animals. Exp. Anim. Tokyo 42, 1–9 (1993).

  45. 45.

    Schenck, L. P., Surette, M. G. & Bowdish, D. M. Composition and immunological significance of the upper respiratory tract microbiota. FEBS Lett. 590, 3705–3720 (2016).

  46. 46.

    Brisse, S. et al. wzi gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J. Clin. Microbiol. 51, 4073–4078 (2013).

  47. 47.

    Deshmukh, H. S. et al. The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice. Nat. Med. 20, 524–530 (2014).

  48. 48.

    Redhu, N. S. et al. Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor. eLife 6, e27652 (2017).

  49. 49.

    Clarke, T. B. Early innate immunity to bacterial infection in the lung is regulated systemically by the commensal microbiota via nod-like receptor ligands. Infect. Immun. 82, 4596–4606 (2014).

  50. 50.

    Mullish, B. H. et al. Functional microbiomics: evaluation of gut microbiota-bile acid metabolism interactions in health and disease. Methods 149, 49–58 (2018).

  51. 51.

    Callahan, B. J. et al. DADA2: high-resolution sample inference from illumina amplicon data. Nat. Methods 13, 581–583 (2016).

Download references

Acknowledgements

T.B.C. is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and Royal Society (grant no. 107660/Z/15Z). For J.A.K.M. and J.R.M., the Division of Integrative Systems Medicine and Digestive Disease at Imperial College London receives financial support from the National Institute for Health Research Imperial Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. We thank Dr A. Clements (Imperial College London) for providing K. pneumoniae strains, G. Donaldson and S. Mazmanian (California Institute of Technology) for providing WT and ΔCCF B. fragilis and B. vulgatus strains, and R. Brown and M. Larkinson (Imperial College London) for providing macrophages.

Author information

T.B.C. and R.P.S. conceived and performed all colonization, transmission and immunological experiments, analysed data and wrote the manuscript. J.A.K.M. and J.R.M. performed sequencing experiments, analysed sequencing data and contributed to the manuscript. J.R.M. and T.B.C. acquired funding.

Correspondence to Thomas B. Clarke.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Bacteroidetes protect against K. pneumoniae in the intestine.

Experimental scheme: “ABX” = MNVA in drinking water; “CC” = commensal consortia inoculation by oral gavage (see Materials and Methods); “Kp” = K. pneumoniae oral inoculation; and “Sample” = fecal sampling day. Days relative to K. pneumoniae inoculation. Mice were orally inoculated with alternative intestinal Bacteroidetes consortium (5×108 CFU B. dorei and B. finegoldi) or Firmicutes (5×108 CFU L. reuteri and L. johnsonii) consortium (n=5 animals). Statistical comparison was by Mann-Whitney (two-tailed), horizontal lines indicate median values. Source data

Extended Data Fig. 2 Bacterial load and composition in feces of non-antibiotic treated, antibiotic-treated and commensal consortia inoculated mice.

Experimental mouse groups are indicated on the left-hand side. From top to bottom, “–ABX” are mice before experimental manipulations; “+ABX” are mice administered MNVA antibiotics in drinking water; “+Microbiota” are mice that have been administered MNVA antibiotics in drinking water and then transferred intestinal and upper airway microbiota; the remaining groups (for example “+Bacteroidetes”) are mice administered MNVA antibiotics in drinking water and then orally administered the indicated commensal consortium (as in Fig. 2). For all of these groups, from left to right, the data are: total 16s rRNA gene copies in adult mouse feces; relative abundance of bacterial taxa in mouse feces (each bar represents the means of n=5 animals); and 16s rRNA gene copies in adult mouse feces of indicated commensal consortia (for example, in the mice administered the “+Bacteroidetes”, “Consortia colonization” levels are the 16s rRNA gene copies for Bacteroidetes in those mice). Statistical comparisons were by Kruskal–Wallis test with Dunn’s multiple comparison test, vertical lines indicate median values.

Extended Data Fig. 3 Bacteroidetes protect against Klebsiella pneumoniae colonization in the intestine through immune signalling.

a, Experimental scheme for (b): “Antibiotics” = MNVA in drinking water; “MT” = microbiota transfer; “Kp” = K. pneumoniae oral inoculation; and “Sample” = fecal sampling day; “Dexa” = dexamethasone treatment. Days relative to K. pneumoniae inoculation. b, K. pneumoniae (OXA-48) burden in the feces of adult mice (n=6,7 animals). c,d,e): acronyms as in (a) with “CC” = commensal consortia inoculation by oral gavage (see Materials and Methods). (d,e) K. pneumoniae (OXA-48) burden (n=8 animals) (d) and K. pneumoniae (ST258) (n=6 animals) (e) in the feces of adult mice. (f) Experimental plan for (g): acronyms as in (a,c). (g) K. pneumoniae (OXA-48) burden (n=7,8 animals) in the feces of adult mice. All statistical comparisons were made by Mann-Whitney test (two-tailed), horizontal lines indicate median values, ND is none detected (limit of detection for K. pneumoniae in feces = 103 CFU/g). Source data

Extended Data Fig. 4 IL-36γ promotes killing of K. pneumoniae by macrophages.

Bone marrow derived-macrophages (a,b) and J774 macrophages (c,d) were incubated with K. pneumoniae ST258 (a,c) and OXA-48 (b,d) (at an MOI of 1:10) for 3 hours. Indicated groups were treated with recombinant IL-36γ (50 ng/ml) for 3 hours prior to infection. Bacterial viability was then determined and expressed relative to the initial number of bacteria in the assay (n=5 independent determinations). Statistical comparisons were by Student’s t-test (two-tailed), error bars are standard deviation, bars represent mean values.

Extended Data Fig. 5 The microbiota primes upper airway immunity but encapsulation allows Klebsiella pneumoniae to overcome these defences.

a, Experimental scheme for (b): “ABX” = MNVA in drinking water; “MT” = microbiota transfer; “Kp” = K. pneumoniae intranasal inoculation; and “Sample” = upper airway lavage sampling; “Dexa” = dexamethasone treatment. Days relative to K. pneumoniae inoculation. Upper airway cytokine levels in adult mice inoculated with K. pneumoniae (WT B5055), unencapsulated K. pneumoniae (∆CPS B5055), and complemented K. pneumoniae (∆CPS::CPS B5055) (n=5,7 animals). Horizontal lines indicate median values, error bars are standard deviation. (c) Upper airway commensal levels in adult mice on the final day of dexamethasone administration (n=5 animals). Statistical comparisons were made Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Horizontal lines indicate median values.

Extended Data Fig. 6 Bacterial load and composition in upper airways and lungs of non-antibiotic treated, antibiotic-treated and commensal consortia inoculated mice.

a, Total bacterial load and composition in adult mouse lung. Relative abundance of bacterial phyla adult mouse lung (each bar represents the means of n=5 animal). Experimental mouse groups are indicated on the left-hand side. From top to bottom, “–ABX” are mice before experimental manipulations; “+ABX” are mice which have been administered MNVA antibiotics in drinking water; “+Microbiota” are mice that have been administered MNVA antibiotics in drinking water and then transferred intestinal and upper airway microbiota. b, Total bacterial load and composition in adult mouse upper airway. Experimental mouse groups as in (a) with addition of consortia inoculated groups (for example “+Bacteroidetes”). These are mice administered MNVA antibiotics in drinking water and then intransally administered the indicated commensal consortium (as in Fig. 6). a,b, From left to right the data are: total 16s rRNA gene copies in upper airway lavage fluid or lung; relative abundance of bacterial phyla in upper airway lavage fluid or lung (each bar represents the means of n=5 animals); and 16s rRNA gene copies in upper airway lavage fluid from mice inoculated with indicated commensal consortia (for example, in the mice administered the “+Bacteroidetes”, “Consortia colonization” levels are the 16s rRNA gene copies for Bacteroidetes those mice). Statistical comparisons were by Kruskal–Wallis test with Dunn’s multiple comparison test, vertical lines indicate median values.

Extended Data Fig. 7 The neonatal microbiota does not protect against encapsulated or unencapsulated K. pneumoniae.

a, Upper airway burden of K. pneumoniae (WT B5055) and unencapsulated K. pneumoniae (∆CPS B5055) in neonatal mice 1 day post-intranasal inoculation (n=4 animals). b, Upper airway cytokine levels in neonatal mice 1 day post-intranasal inoculation with K. pneumoniae (n=5 animals). Statistical comparisons were made by Kruskal–Wallis test with Dunn’s correction for multiple comparisons, horizontal lines indicate median values, error bars are standard deviation. Source data

Extended Data Fig. 8 Proteobacteria restore homeostatic upper airway IL-17A production and K. pneumoniae-induced TNF production in the upper airway during colonization.

a, Experimental scheme for (b,c): “ABX” = MNVA in drinking water; “CC” = commensal consortia inoculation by oral gavage (see Materials and Methods); “Kp” = K. pneumoniae oral inoculation; and “Sample” = upper airway sampling day. Days relative to K. pneumoniae inoculation. b, Upper airway TNF levels in adult mice 1 day post-intranasal inoculation with K. pneumoniae (WT B5055) and unencapsulated K. pneumoniae (∆CPS B5055) (n=5 animals). c, Homeostatic upper airway IL-17A levels in adult mice on day 0 (without K. pneumoniae inoculation) (n=5). Statistical comparisons were made by Kruskal–Wallis test with Dunn’s correction for multiple comparisons, horizontal lines indicate median values, error bars are standard deviation.

Extended Data Fig. 9 Antibiotic resistance profiles of K. pneumoniae in contact mice matches antibiotic resistance profile of K. pneumoniae administered to index mice.

a, Fecal K. penumoniae burden in samples from neonatal contact mice before and after cohousing with K. penumoniae OXA-48 (resistant to azithromycin and ampicillin and sensitive to kanamycin) colonized index mice (n=6,7 animals) (a). b,c Fecal K. penumoniae burden in samples from adult contact mice before and after cohousing with K. penumoniae OXA-48 colonized index mice (n=6,7 animals) (b) or K. penumoniae ST258 (resistant to kanamycin and ampicillin and sensitive to gentamycin) colonized index mice (n=6,7 animals) (c). Culture media was supplemented with kanamycin, ampicillin, gentamycin and azithromycin were indicated (at 50 μg/mL). ND is none detected (the limit of detection for K. pneumoniae in feces = 103 CFU/g), horizontal lines indicate mean values. Source data

Extended Data Fig. 10 Intestinal Bacteroidetes protect against K. pneumoniae transmission via IL-36 and this requires the Bacteroidetes commensal colonization factors.

Summary of K. pneumoniae transmission in neonatal and adult mice. In index mice, “+Antibiotics” in the microbiota manipulation column indicates azithromycin and cefotaxime treatment to establish K. pneumoniae colonization. Bacteroidetes and Firmicutes consortia used as in Fig. 2. Anti-IL-36R treatment as in Fig. 4. All experiments were with the K. pneumoniae OXA-48, except 7, 9, 11, 14, 15, and 18 with K. pneumoniae ST258. Successful transmission to the upper airway was defined as the presence of any detectable K. pneumoniae in the upper airway of contact mice after 4 days cohousing. Successful transmission to the intestine was defined as the presence of >103 CFU K. pneumoniae/g of feces (the limit of detection for K. pneumoniae in feces) of contact mice after 4 days cohousing. Statistical significance calculated using Fisher’s exact test (two-sided).

Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and Supplementary Tables 1 and 2.

Reporting Summary

Source data

Source Data Fig. 1

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Fig. 2

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Fig. 3

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Fig. 4

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Fig. 5

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Extended Data Fig. 1

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Extended Data Fig. 3

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Extended Data Fig. 7

Statistical Source Data of animal experiments modelling bacterial colonization.

Source Data Extended Data Fig. 9

Statistical Source Data of animal experiments modelling bacterial colonization.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sequeira, R.P., McDonald, J.A.K., Marchesi, J.R. et al. Commensal Bacteroidetes protect against Klebsiella pneumoniae colonization and transmission through IL-36 signalling. Nat Microbiol 5, 304–313 (2020). https://doi.org/10.1038/s41564-019-0640-1

Download citation