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Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection

Nature Medicine volume 22, pages 1330–1334 (2016)Cite this article

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An Erratum to this article was published on 06 December 2016

This article has been updated

Abstract

Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and is an urgent public health concern worldwide1. Over the past decade, incidence, severity and costs associated with C. difficile infection (CDI) have increased dramatically2. CDI is most commonly initiated by antibiotic-mediated disruption of the gut microbiota; however, non-antibiotic-associated CDI cases are well documented and on the rise3,4. This suggests that unexplored environmental, nutrient and host factors probably influence CDI. Here we show that excess dietary zinc (Zn) substantially alters the gut microbiota and, in turn, reduces the minimum amount of antibiotics needed to confer susceptibility to CDI. In mice colonized with C. difficile, excess dietary Zn severely exacerbated C. difficile–associated disease by increasing toxin activity and altering the host immune response. In addition, we show that the Zn-binding S100 protein calprotectin has antimicrobial effects against C. difficile and is an essential component of the innate immune response to CDI. Taken together, these data suggest that nutrient Zn levels have a key role in determining susceptibility to CDI and severity of disease, and that calprotectin-mediated metal limitation is an important factor in the host immune response to C. difficile.

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Figure 1: Increased dietary Zn alters tissue Zn levels and dramatically alters the gut microbiota.
Figure 2: Excess dietary Zn exacerbates C. difficile–associated disease.
Figure 3: Excess dietary Zn decreases the threshold of antibiotics needed to confer susceptibility to CDI.
Figure 4: Calprotectin is essential for combating CDI.

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  • 20 October 2016

    In the version of this article initially published online, the low-zinc diet was incorrectly described as 0 mg per kg body weight (mg/kg) Zn and should be described as 'low-Zn diet; 0 mg Zn per kg of diet (mg/kg)'. The error has been corrected in the print, PDF and HTML versions of this article.

References

  1. Lessa, F.C. et al. Burden of Clostridium difficile infection in the United States. N. Engl. J. Med. 372, 825–834 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Kelly, C.P. & LaMont, J.T. Clostridium difficile—more difficult than ever. N. Engl. J. Med. 359, 1932–1940 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Theriot, C.M. et al. Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection. Nat. Commun. 5, 3114 (2014).

    Article  PubMed  CAS  Google Scholar 

  4. Rupnik, M., Wilcox, M.H. & Gerding, D.N. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat. Rev. Microbiol. 7, 526–536 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Leffler, D.A. & Lamont, J.T. Clostridium difficile infection. N. Engl. J. Med. 373, 287–288 (2015).

    CAS  PubMed  Google Scholar 

  6. Gerding, D.N., Muto, C.A. & Owens, R.C. Jr. Measures to control and prevent Clostridium difficile infection. Clin. Infect. Dis. 46 (Suppl. 1), S43–S49 (2008).

    Article  PubMed  Google Scholar 

  7. Fekety, R. et al. Epidemiology of antibiotic-associated colitis; isolation of Clostridium difficile from the hospital environment. Am. J. Med. 70, 906–908 (1981).

    Article  CAS  PubMed  Google Scholar 

  8. Schubert, A.M., Sinani, H. & Schloss, P.D. Antibiotic-induced alterations of the murine gut microbiota and subsequent effects on colonization resistance against Clostridium difficile. MBio 6, e00974 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Buffie, C.G. et al. Precision microbiome reconstitution restores bile-acid-mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. David, L.A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Fischer Walker, C. & Black, R.E. Zinc and the risk for infectious disease. Annu. Rev. Nutr. 24, 255–275 (2004).

    Article  PubMed  CAS  Google Scholar 

  12. Hood, M.I. & Skaar, E.P. Nutritional immunity: transition metals at the pathogen–host interface. Nat. Rev. Microbiol. 10, 525–537 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Turnbaugh, P.J. et al. The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci. Transl. Med. 1, 6ra14 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Chumbler, N.M. et al. Crystal structure of Clostridium difficile toxin A. Nature Microbiology 1, 15002 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cafardi, V. et al. Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins. PLoS One 8, e81306 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Rubino, J.T. et al. Structural characterization of zinc-bound Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile. J. Biol. Inorg. Chem. 21, 185–196 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Hensbergen, P.J. et al. Clostridium difficile secreted Pro–Pro endopeptidase PPEP-1 (ZMP1 or CD2830) modulates adhesion through cleavage of the collagen-binding protein CD2831. FEBS Lett. 589 24 Pt B, 3952–3958 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Theriot, C.M. et al. Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains. Gut Microbes 2, 326–334 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Wüst, J., Sullivan, N.M., Hardegger, U. & Wilkins, T.D. Investigation of an outbreak of antibiotic-associated colitis by various typing methods. J. Clin. Microbiol. 16, 1096–1101 (1982).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Stabler, R.A. et al. Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol. 10, R102 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Hasegawa, M. et al. Protective role of commensals against Clostridium difficile infection via an IL-1β-mediated positive-feedback loop. J. Immunol. 189, 3085–3091 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Seo, S.U. et al. Distinct commensals induce interleukin-1β via NLRP3 inflammasome in inflammatory monocytes to promote intestinal inflammation in response to injury. Immunity 42, 744–755 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zackular, J.P., Chazin, W.J. & Skaar, E.P. Nutritional immunity: S100 proteins at the host–pathogen interface. J. Biol. Chem. 290, 18991–18998 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hood, M.I. et al. Identification of an Acinetobacter baumannii zinc acquisition system that facilitates resistance to calprotectin-mediated zinc sequestration. PLoS Pathog. 8, e1003068 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kehl-Fie, T.E. et al. Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus. Cell Host Microbe 10, 158–164 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gaddy, J.A. et al. The host protein calprotectin modulates the Helicobacter pylori cag type IV secretion system via zinc sequestration. PLoS Pathog. 10, e1004450 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Hanania, A. et al. Fecal calprotectin in the diagnosis of Clostridium difficile infection. Infect. Dis. Clin. Pract. (Baltim. Md.) 24, 31–34 (2016).

    Article  Google Scholar 

  28. Rao, K., Santhosh, K., Mogle, J.A., Higgins, P.D. & Young, V.B. Elevated fecal calprotectin associates with adverse outcomes from Clostridium difficile infection in older adults. Infect. Dis. (Lond.) 48, 663–669 (2016).

    Article  CAS  Google Scholar 

  29. Hobbs, J.A. et al. Myeloid cell function in MRP-14 (S100A9)-null mice. Mol. Cell. Biol. 23, 2564–2576 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Reeves, P.G., Nielsen, F.H. & Fahey, G.C. Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951 (1993).

    Article  CAS  PubMed  Google Scholar 

  31. Reeves, A.E. et al. The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile infection. Gut Microbes 2, 145–158 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kehl-Fie, T.E. et al. MntABC and MntH contribute to systemic Staphylococcus aureus infection by competing with calprotectin for nutrient manganese. Infect. Immun. 81, 3395–3405 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zackular, J.P., Rogers, M.A., Ruffin, M.T. IV & Schloss, P.D. The human gut microbiome as a screening tool for colorectal cancer. Cancer Prev. Res. (Phila.) 7, 1112–1121 (2014).

    Article  CAS  Google Scholar 

  34. Schloss, P.D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Pruesse, E. et al. SILVA: a comprehensive online resource for quality-checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang, Q., Garrity, G.M., Tiedje, J.M. & Cole, J.R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Magurran, A.E. & Henderson, P.A. Temporal turnover and the maintenance of diversity in ecological assemblages. Phil. Trans. R. Soc. Lond. B 365, 3611–3620 (2010).

    Article  Google Scholar 

  39. Yue, J.C. & Clayton, M.K. A similarity measure based on species proportions. Commun. Stat. Theory Methods 34, 2123–2131 (2005).

    Article  Google Scholar 

  40. Vaishnava, S. et al. The antibacterial lectin Reg-IIIã promotes the spatial segregation of microbiota and host in the intestine. Science 334, 255–258 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Damo, S.M. et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc. Natl. Acad. Sci. USA 110, 3841–3846 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Siew, E.D. et al. Urine neutrophil gelatinase-associated lipocalin moderately predicts acute kidney injury in critically ill adults. J. Am. Soc. Nephrol. 20, 1823–1832 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Martin, A.P. Phylogenetic approaches for describing and comparing the diversity of microbial communities. Appl. Environ. Microbiol. 68, 3673–3682 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Cutler, D.R. et al. Random forests for classification in ecology. Ecology 88, 2783–2792 (2007).

    Article  PubMed  Google Scholar 

  45. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple-testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).

    Google Scholar 

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Acknowledgements

We thank P. Schloss and J. Sorg for critical feedback on this study, and D. Aronoff and S. Walk for providing C. difficile strains. This research was supported by the US Department of Veterans Affairs (Merit Review Award no. 1I01BX002482; E.P.S.), the US National Institutes of Health (NIH) (grant no. R01 AI101171 (E.P.S.) and P41 GM103391-05 (R.M.C.)) and the Vanderbilt Digestive Disease Research Center (VDDRC) (grant no. P30DK058404; E.P.S.). J.P.Z. was supported by NIH–NIDDK grant no. T32DK007673 and NIH–NIAID grant no. F32AI120553. J.L.M. was supported by NIH–NIGMS grant no. T32GM065086. M.R.N. was supported by the Thrasher Research Fund Early Career Award.

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Authors and Affiliations

  1. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Joseph P Zackular, Ashley T Jordan, Lillian J Juttukonda, Michael J Noto, Yaofang Zhang, Lorraine B Ware, M Kay Washington & Eric P Skaar

  2. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    Jessica L Moore, Walter J Chazin & Richard M Caprioli

  3. Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, USA

    Jessica L Moore & Richard M Caprioli

  4. Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Michael J Noto, Matthew W Semler & Lorraine B Ware

  5. Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Maribeth R Nicholson

  6. Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

    Jonathan D Crews

  7. Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA

    Walter J Chazin & Richard M Caprioli

  8. Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA

    Walter J Chazin

  9. Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Richard M Caprioli

  10. US Department of Veterans Affairs, Tennessee Valley Healthcare Systems, Nashville, Tennessee, USA

    Eric P Skaar

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Contributions

J.P.Z. and E.P.S. designed experiments and wrote the manuscript with input from the co-authors; J.P.Z. performed animal experiments, the corresponding assays and analyses with assistance from A.T.J.; J.P.Z. performed microbiota community analyses; L.J.J. designed altered metal diets; J.L.M. and R.M.C. performed MALDI–MS imaging and corresponding analyses; Y.Z. and R.M.C. performed ICP–MS; M.K.W. performed histological analyses; W.J.C. assisted in calprotectin assays; M.R.N. and J.D.C. enlisted pediatric patients and collected fecal samples; M.W.S., M.J.N. and L.B.W. collected adult serum and aided in the analyses of calprotectin experiments.

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Correspondence to Eric P Skaar.

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Zackular, J., Moore, J., Jordan, A. et al. Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection. Nat Med 22, 1330–1334 (2016). https://doi.org/10.1038/nm.4174

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