1887

Abstract

is an emerging nosocomial pathogen and one of the major causes of antibiotic-associated diarrhoea. Cases of -associated disease (CDAD) are likely initiated by the ingestion of dormant spores, which then germinate, outgrow and rapidly proliferate to cause gastrointestinal (GI) infections. To understand the initial stages of CDAD pathogenesis, we have characterized the germination of spores from a collection of strains, including some clinical isolates obtained from a CDAD outbreak (CDAD isolates). Spores of one laboratory strain and five CDAD isolates did not germinate with amino acids, but did germinate on a nutrient-rich medium. However, bile salts had little effect on spore germination, either alone or in a nutrient-rich medium. These spores also germinated with KCl, as well as the non-nutrient germinants dodecylamine and a 1 : 1 chelate of Ca and dipicolinic acid. An unexpected finding was that spores of most of the strains also germinated with inorganic phosphate (P) with a pH optimum of 6. The germination of spores of CDAD strains with KCl and P, two molecules present at significant levels in the GI tract, suggests that spores germinate in the human body by sensing P in the early segments of the duodenum and KCl in the colon.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/016592-0
2008-08-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/8/2241.html?itemId=/content/journal/micro/10.1099/mic.0.2008/016592-0&mimeType=html&fmt=ahah

References

  1. Ando, Y. ( 1974; ). Ionic germination of spores of Clostridium perfringens type A. Jpn J Microbiol 18, 433–439.[CrossRef]
    [Google Scholar]
  2. Bartlett, J. G. ( 1992; ). Antibiotic-associated diarrhea. Clin Infect Dis 15, 573–581.[CrossRef]
    [Google Scholar]
  3. Bean, N. H., Griffin, P. M., Goulding, J. S. & Ivey, C. B. ( 1990; ). Foodborne disease outbreaks, 5-year summary, 1983–1987. MMWR CDC Surveill Summ 39, 15–57.
    [Google Scholar]
  4. Borriello, S. P. ( 1998; ). Pathogenesis of Clostridium difficile infection. J Antimicrob Chemother 41 (Suppl. C), 13–19.
    [Google Scholar]
  5. Borriello, S. P., Davies, H. A., Kamiya, S., Reed, P. J. & Seddon, S. ( 1990; ). Virulence factors of Clostridium difficile. Rev Infect Dis 12 (Suppl. 2), S185–S191.[CrossRef]
    [Google Scholar]
  6. Broussolle, V., Alberto, F., Shearman, C. A., Mason, D. R., Botella, L., Nguyen-The, C., Peck, M. W. & Carlin, F. ( 2002; ). Molecular and physiological characterization of spore germination in Clostridium botulinum and C. sporogenes. Anaerobe 8, 89–100.[CrossRef]
    [Google Scholar]
  7. Cabrera-Martinez, R. M., Tovar-Rojo, F., Vepachedu, V. R. & Setlow, P. ( 2003; ). Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. J Bacteriol 185, 2457–2464.[CrossRef]
    [Google Scholar]
  8. Christie, G. & Lowe, C. R. ( 2007; ). Role of chromosomal and plasmid-borne receptor homologues in the response of Bacillus megaterium QM B1551 spores to germinants. J Bacteriol 189, 4375–4383.[CrossRef]
    [Google Scholar]
  9. Clements, M. O. & Moir, A. ( 1998; ). Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180, 6729–6735.
    [Google Scholar]
  10. Cortezzo, D. E., Setlow, B. & Setlow, P. ( 2004; ). Analysis of the action of compounds that inhibit the germination of spores of Bacillus species. J Appl Microbiol 96, 725–741.[CrossRef]
    [Google Scholar]
  11. Cowan, A. E., Koppel, D. E., Setlow, B. & Setlow, P. ( 2003; ). A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy. Proc Natl Acad Sci U S A 100, 4209–4214.[CrossRef]
    [Google Scholar]
  12. Duncan, C. L. & Strong, D. H. ( 1968; ). Improved medium for sporulation of Clostridium perfringens. Appl Microbiol 16, 82–89.
    [Google Scholar]
  13. Foster, S. J. & Johnstone, K. ( 1987; ). Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM. Biochem J 242, 573–579.
    [Google Scholar]
  14. Hurley, B. W. & Nguyen, C. C. ( 2002; ). The spectrum of pseudomembranous enterocolitis and antibiotic-associated diarrhea. Arch Intern Med 162, 2177–2184.[CrossRef]
    [Google Scholar]
  15. Ishikawa, S., Yamane, K. & Sekiguchi, J. ( 1998; ). Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores. J Bacteriol 180, 1375–1380.
    [Google Scholar]
  16. Johnson, L. R. ( 2000; ). Gastrointestinal Physiology, 6th edn. St Louis, MO: Mosby, Inc.
  17. Johnson, S., Clabots, C. R., Linn, F. V., Olson, M. M., Peterson, L. R. & Gerding, D. N. ( 1990; ). Nosocomial Clostridium difficile colonisation and disease. Lancet 336, 97–100.[CrossRef]
    [Google Scholar]
  18. Jump, R. L., Pultz, M. J. & Donskey, C. J. ( 2007; ). Vegetative Clostridium difficile survives in room air on moist surfaces and in gastric contents with reduced acidity: a potential mechanism to explain the association between proton pump inhibitors and C. difficile-associated diarrhea? Antimicrob Agents Chemother 51, 2883–2887.[CrossRef]
    [Google Scholar]
  19. Kelly, C. P. & LaMontt, J. T. ( 1998; ). Clostridium difficile infection. Annu Rev Med 49, 375–390.[CrossRef]
    [Google Scholar]
  20. Kelly, C. P., Pothoulakis, C. & LaMont, J. T. ( 1994; ). Clostridium difficile colitis. N Engl J Med 330, 257–262.[CrossRef]
    [Google Scholar]
  21. Kokai-Kun, J. F., Songer, J. G., Czeczulin, J. R., Chen, F. & McClane, B. A. ( 1994; ). Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol 32, 2533–2539.
    [Google Scholar]
  22. Kumazawa, T., Masayama, A., Fukuoka, S., Makino, S., Yoshimura, T. & Moriyama, R. ( 2007; ). Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40. Biosci Biotechnol Biochem 71, 884–892.[CrossRef]
    [Google Scholar]
  23. Kyne, L., Hamel, M. B., Polavaram, R. & Kelly, C. P. ( 2002; ). Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 34, 346–353.[CrossRef]
    [Google Scholar]
  24. Lemann, J. ( 1993; ). Intestinal absorption of calcium, magnesium, and phosphorus. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, pp. 46–50. Edited by M. J. Favus. New York: The American Society for Bone and Mineral Research.
  25. Makino, S., Ito, N., Inoue, T., Miyata, S. & Moriyama, R. ( 1994; ). A spore-lytic enzyme released from Bacillus cereus spores during germination. Microbiology 140, 1403–1410.[CrossRef]
    [Google Scholar]
  26. McEllistrem, M. C., Carman, R. J., Gerding, D. N., Genheimer, C. W. & Zheng, L. ( 2005; ). A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes. Clin Infect Dis 40, 265–272.[CrossRef]
    [Google Scholar]
  27. McFarland, L. V., Mulligan, M. E., Kwok, R. Y. & Stamm, W. E. ( 1989; ). Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 320, 204–210.[CrossRef]
    [Google Scholar]
  28. Miyata, S., Moriyama, R., Sugimoto, K. & Makino, S. ( 1995; ). Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores. Biosci Biotechnol Biochem 59, 514–515.[CrossRef]
    [Google Scholar]
  29. Miyata, S., Kozuka, S., Yasuda, Y., Chen, Y., Moriyama, R., Tochikubo, K. & Makino, S. ( 1997; ). Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy. FEMS Microbiol Lett 152, 243–247.[CrossRef]
    [Google Scholar]
  30. Moir, A., Corfe, B. M. & Behravan, J. ( 2002; ). Spore germination. Cell Mol Life Sci 59, 403–409.[CrossRef]
    [Google Scholar]
  31. Myers, G. S., Rasko, D. A., Cheung, J. K., Ravel, J., Seshadri, R., DeBoy, R. T., Ren, Q., Varga, J., Awad, M. M. & other authors ( 2006; ). Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16, 1031–1040.[CrossRef]
    [Google Scholar]
  32. Nakamura, S., Yamakawa, K., Izumi, J., Nakashio, S. & Nishida, S. ( 1985; ). Germinability and heat resistance of spores of Clostridium difficile strains. Microbiol Immunol 29, 113–118.[CrossRef]
    [Google Scholar]
  33. O'Connor, J. R., Lyras, D., Farrow, K. A., Adams, V., Powell, D. R., Hinds, J., Cheung, J. K. & Rood, J. I. ( 2006; ). Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol 61, 1335–1351.[CrossRef]
    [Google Scholar]
  34. Paidhungat, M. & Setlow, P. ( 2000; ). Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182, 2513–2519.[CrossRef]
    [Google Scholar]
  35. Paidhungat, M., Setlow, B., Driks, A. & Setlow, P. ( 2000; ). Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182, 5505–5512.[CrossRef]
    [Google Scholar]
  36. Paidhungat, M., Ragkousi, K. & Setlow, P. ( 2001; ). Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J Bacteriol 183, 4886–4893.[CrossRef]
    [Google Scholar]
  37. Paidhungat, M., Setlow, B., Daniels, W. B., Hoover, D., Papafragkou, E. & Setlow, P. ( 2002; ). Mechanisms of induction of germination of Bacillus subtilis spores by high pressure. Appl Environ Microbiol 68, 3172–3175.[CrossRef]
    [Google Scholar]
  38. Paredes-Sabja, D., Sarker, N., Setlow, B., Setlow, P. & Sarker, M. R. ( 2008a; ). Roles of DacB and Spm proteins in Clostridium perfringens spore resistance to moist heat, chemicals and UV radiation. Appl Environ Microbiol 74, 3730–3738.[CrossRef]
    [Google Scholar]
  39. Paredes-Sabja, D., Setlow, B., Setlow, P. & Sarker, M. R. ( 2008b; ). Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid. J Bacteriol 190, 4648–4659.[CrossRef]
    [Google Scholar]
  40. Paredes-Sabja, D., Torres, J. A., Setlow, P. & Sarker, M. R. ( 2008c; ). Clostridium perfringens spore germination: characterization of germinants and their receptors. J Bacteriol 190, 1190–1201.[CrossRef]
    [Google Scholar]
  41. Philippe, V. A., Mendez, M. B., Huang, I. H., Orsaria, L. M., Sarker, M. R. & Grau, R. R. ( 2006; ). Inorganic phosphate induces spore morphogenesis and enterotoxin production in the intestinal pathogen Clostridium perfringens. Infect Immun 74, 3651–3656.[CrossRef]
    [Google Scholar]
  42. Rode, L. J. & Foster, J. W. ( 1962; ). Ionic germination of spores of Bacillus megaterium QM B 1551. Arch Mikrobiol 43, 183–200.[CrossRef]
    [Google Scholar]
  43. Rotman, Y. & Fields, M. L. ( 1968; ). A modified reagent for dipicolinic acid analysis. Anal Biochem 22, 168 [CrossRef]
    [Google Scholar]
  44. Rune, S. J. ( 1973; ). pH in the human duodenum. Its physiological and pathophysiological significance. Digestion 8, 261–268.[CrossRef]
    [Google Scholar]
  45. Sebaihia, M., Wren, B. W., Mullany, P., Fairweather, N. F., Minton, N., Stabler, R., Thomson, N. R., Roberts, A. P., Cerdeño-Tárraga, A. M. & other authors ( 2006; ). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38, 779–786.[CrossRef]
    [Google Scholar]
  46. Setlow, P. ( 2003; ). Spore germination. Curr Opin Microbiol 6, 550–556.[CrossRef]
    [Google Scholar]
  47. Setlow, P. ( 2006; ). Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514–525.[CrossRef]
    [Google Scholar]
  48. Setlow, B., Cowan, A. E. & Setlow, P. ( 2003; ). Germination of spores of Bacillus subtilis with dodecylamine. J Appl Microbiol 95, 637–648.[CrossRef]
    [Google Scholar]
  49. Shimamoto, S., Moriyama, R., Sugimoto, K., Miyata, S. & Makino, S. ( 2001; ). Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity. J Bacteriol 183, 3742–3751.[CrossRef]
    [Google Scholar]
  50. Sorg, J. A. & Sonenshein, A. L. ( 2008; ). Bile salts and glycine as co-germinants for Clostridium difficile spores. J Bacteriol 190, 2505–2512.[CrossRef]
    [Google Scholar]
  51. Vepachedu, V. R. & Setlow, P. ( 2005; ). Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis. J Bacteriol 187, 5677–5682.[CrossRef]
    [Google Scholar]
  52. Vepachedu, V. R. & Setlow, P. ( 2007; ). Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme. J Bacteriol 189, 1565–1572.[CrossRef]
    [Google Scholar]
  53. Voth, D. E. & Ballard, J. D. ( 2005; ). Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18, 247–263.[CrossRef]
    [Google Scholar]
  54. Walton, J. & Gray, T. K. ( 1979; ). Absorption of inorganic phosphate in the human small intestine. Clin Sci (Lond) 56, 407–412.
    [Google Scholar]
  55. Wilcox, M. H., Fawley, W. N. & Parnell, P. ( 2000; ). Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery of Clostridium difficile. J Hosp Infect 44, 65–69.[CrossRef]
    [Google Scholar]
  56. Wilson, K. H. ( 1983; ). Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J Clin Microbiol 18, 1017–1019.
    [Google Scholar]
  57. Wilson, K. H., Kennedy, M. J. & Fekety, F. R. ( 1982; ). Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J Clin Microbiol 15, 443–446.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/016592-0
Loading
/content/journal/micro/10.1099/mic.0.2008/016592-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error