1887

Abstract

The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, and . Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone–yeast extract–glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm had increased by 30–35 %. With at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/016576-0
2008-07-01
2019-11-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/7/1927.html?itemId=/content/journal/micro/10.1099/mic.0.2008/016576-0&mimeType=html&fmt=ahah

References

  1. Amy, P. S. & Morita, R. Y. ( 1983; ). Starvation-survival patterns of sixteen freshly isolated open-ocean bacteria. Appl Environ Microbiol 45, 1109–1115.
    [Google Scholar]
  2. Bhupathiraju, V. K., Hernandez, M., Landfear, D. & Alvarez-Cohen, L. ( 1999; ). Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria. J Microbiol Methods 37, 231–243.[CrossRef]
    [Google Scholar]
  3. Bowden, G. H. ( 1991; ). Which bacteria are cariogenic in humans? In Dental Caries, pp. 266–286. Edited by N. W. Johnson. Cambridge: Cambridge University Press.
  4. Bowden, G. H. W. ( 1999; ). Controlled environment model for accumulation of biofilms of oral bacteria. Methods Enzymol 310, 216–224.
    [Google Scholar]
  5. Bowden, G. H. & Hamilton, I. R. ( 1989; ). Competition between Streptococcus mutans and Lactobacillus casei in mixed continuous culture. Oral Microbiol Immunol 4, 57–64.[CrossRef]
    [Google Scholar]
  6. Bowden, G. H. & Hamilton, I. R. ( 1998; ). Survival of oral bacteria. Crit Rev Oral Biol Med 9, 54–85.[CrossRef]
    [Google Scholar]
  7. Bowden, G. H. & Li, Y. H. ( 1997; ). Nutritional influences on biofilm development. Adv Dent Res 11, 81–99.[CrossRef]
    [Google Scholar]
  8. Brecx, M., Netuschil, L., Reichert, B. & Schereil, G. ( 1990; ). Efficacy of Listerine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J Clin Periodontol 17, 292–297.[CrossRef]
    [Google Scholar]
  9. Brown, M. R. & Smith, A. W. ( 2001; ). Dormancy and persistence in chronic infection: role of the general stress response in resistance to chemotherapy. J Antimicrob Chemother 48, 141–142.[CrossRef]
    [Google Scholar]
  10. Carlsson, J. & Johansson, T. ( 1973; ). Sugar and the production of bacteria in the human mouth. Caries Res 7, 273–282.[CrossRef]
    [Google Scholar]
  11. Chatterji, D. & Ojha, A. K. ( 2001; ). Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 4, 160–165.[CrossRef]
    [Google Scholar]
  12. Chávez de Paz, L. ( 2007; ). Redefining the persistent infection in root canals: possible role of biofilm communities. J Endod 33, 652–662.[CrossRef]
    [Google Scholar]
  13. Chávez de Paz, L. E., Dahlén, G., Molander, A., Möller, Å. & Bergenholtz, G. ( 2003; ). Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment. Int Endod J 36, 500–508.[CrossRef]
    [Google Scholar]
  14. Chávez de Paz, L. E., Molander, A. & Dahlén, G. ( 2004; ). Gram-positive rods prevailing in teeth with apical periodontitis undergoing root canal treatment. Int Endod J 37, 579–587.[CrossRef]
    [Google Scholar]
  15. Chávez de Paz, L., Svensäter, G., Dahlén, G. & Bergenholtz, G. ( 2005; ). Streptococci from root canals in teeth with apical periodontitis receiving endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 100, 232–241.[CrossRef]
    [Google Scholar]
  16. Chávez de Paz, L. E., Bergenholtz, G., Dahlén, G. & Svensäter, G. ( 2007; ). Response to alkaline stress by root canal bacteria in biofilms. Int Endod J 40, 344–355.[CrossRef]
    [Google Scholar]
  17. Davey, M. E. & O'Toole, G. A. ( 2000; ). Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64, 847–867.[CrossRef]
    [Google Scholar]
  18. De Jong, M. H. & Van der Hoeven, J. S. ( 1987; ). The growth of oral bacteria on saliva. J Dent Res 66, 498–505.[CrossRef]
    [Google Scholar]
  19. Foley, I., Marsh, P., Wellington, E. M., Smith, A. W. & Brown, M. R. ( 1999; ). General stress response master regulator rpoS is expressed in human infection: a possible role in chronicity. J Antimicrob Chemother 43, 164–165.[CrossRef]
    [Google Scholar]
  20. Frandsen, E. V., Pedrazzoli, V. & Kilian, M. ( 1991; ). Ecology of viridians streptococci in the oral cavity and pharynx. Oral Microbiol Immunol 6, 129–133.[CrossRef]
    [Google Scholar]
  21. Fukui, M. & Takii, S. ( 1989; ). Reduction of tetrazolium salts by sulfate-reducing bacteria. FEMS Microbiol Ecol 62, 13–19.[CrossRef]
    [Google Scholar]
  22. Giard, J. C., Hartke, A., Flahaut, S., Boutibonnes, P. & Auffray, Y. ( 1997; ). Glucose starvation response in Enterococcus faecalis JH2-2: survival and protein analysis. Res Microbiol 148, 27–35.[CrossRef]
    [Google Scholar]
  23. Gilbert, P., Maira-Litran, T., McBain, A. J., Rickard, A. H. & Whyte, F. W. ( 2002; ). The physiology and collective recalcitrance of microbial biofilm communities. Adv Microb Physiol 46, 202–256.
    [Google Scholar]
  24. Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. ( 2004; ). Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2, 95–108.[CrossRef]
    [Google Scholar]
  25. Holdeman, L. V., Cato, E. P. & Moore, W. E. ( 1977; ). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
  26. Jenkins, D. E., Schultz, J. E. & Matin, A. ( 1988; ). Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli. J Bacteriol 170, 3910–3914.
    [Google Scholar]
  27. Kaprelyants, A. S. & Kell, D. B. ( 1993; ). Dormancy in stationary-phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59, 3187–3196.
    [Google Scholar]
  28. Karner, M. & Fuhrman, J. A. ( 1997; ). Determination of active marine bacterioplankton: a comparison of universal 16S rRNA probes, autoradiography, and nucleoid staining. Appl Environ Microbiol 63, 1208–1213.
    [Google Scholar]
  29. Kim, W. S., Park, J. H., Ren, J., Su, P. & Dunn, N. W. ( 2001; ). Survival response and rearrangement of plasmid DNA of Lactococcus lactis during long-term starvation. Appl Environ Microbiol 67, 4594–4602.[CrossRef]
    [Google Scholar]
  30. Longnecker, K., Sherr, B. F. & Sherr, E. B. ( 2005; ). Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem. Appl Environ Microbiol 71, 7737–7749.[CrossRef]
    [Google Scholar]
  31. Lopez-Amoros, R., Comas, J. & Vives-Rego, J. ( 1995; ). Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol. Appl Environ Microbiol 61, 2521–2526.
    [Google Scholar]
  32. Mah, T. F. & O'Toole, G. A. ( 2001; ). Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9, 34–39.[CrossRef]
    [Google Scholar]
  33. Marsh, P. D. ( 2005; ). Dental plaque: biological significance of a biofilm and community life-style. J Clin Periodontol 32 (Suppl. 6), 7–15.[CrossRef]
    [Google Scholar]
  34. Matin, A. ( 1990; ). Molecular analysis of the starvation stress in Escherichia coli. FEMS Microbiol Lett 74, 185–195.[CrossRef]
    [Google Scholar]
  35. Mechold, U. & Malke, H. ( 1997; ). Characterization of the stringent and relaxed responses of Streptococcus equisimilis. J Bacteriol 179, 2658–2667.
    [Google Scholar]
  36. Miethke, M., Westers, H., Blom, E. J., Kuipers, O. P. & Marahiel, M. A. ( 2006; ). Iron starvation triggers the stringent response and induces amino acid biosynthesis for bacillibactin production in Bacillus subtilis. J Bacteriol 188, 8655–8657.[CrossRef]
    [Google Scholar]
  37. Moat, A. G. & Foster, J. W. ( 1995; ). Amino acids, purines and pyrimidines. In Microbial Physiology, pp. 462–517. Edited by A. G. Moat & J. W. Foster. New York: Wiley-Liss Inc.
  38. Netuschil, L., Reich, E., Unteregger, E., Sculean, A. & Brecx, M. ( 1998; ). A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography. Arch Oral Biol 43, 277–285.[CrossRef]
    [Google Scholar]
  39. Nielsen, J. L., Aquino de Muro, M. & Nielsen, P. H. ( 2003; ). Evaluation of the redox dye 5-cyano-2,3-tolyl-tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization. Appl Environ Microbiol 69, 641–643.[CrossRef]
    [Google Scholar]
  40. Oliver, J. D. ( 1995; ). The viable but non-culturable state in the human pathogen Vibrio vulnificus. FEMS Microbiol Lett 133, 203–208.[CrossRef]
    [Google Scholar]
  41. Rogosa, M., Wiseman, R. F., Mitchell, J. A., Disraely, M. N. & Beaman, A. J. ( 1953; ). Species differentiation of oral lactobacilli from man including description of Lactobacillus salivarius nov spec and Lactobacillus cellobiosus nov spec. J Bacteriol 65, 681–699.
    [Google Scholar]
  42. Ruoff, K. L. ( 1988; ). Streptococcus anginosus (“Streptococcus milleri”): the unrecognized pathogen. Clin Microbiol Rev 1, 102–108.
    [Google Scholar]
  43. Sansone, C., Van Houte, J., Joshipura, K., Kent, R. & Margolis, H. C. ( 1993; ). The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J Dent Res 72, 508–516.[CrossRef]
    [Google Scholar]
  44. Sherr, B. F., del Giorgio, P. & Sherr, E. B. ( 1999; ). Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC. Aquat Microb Ecol 18, 117–131.[CrossRef]
    [Google Scholar]
  45. Smith, J. J. & McFeters, G. A. ( 1997; ). Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12. J Microbiol Methods 29, 161–175.[CrossRef]
    [Google Scholar]
  46. Socransky, S. S. & Haffajee, A. D. ( 2002; ). Dental biofilms: difficult therapeutic targets. Periodontol 2000 28, 12–55.[CrossRef]
    [Google Scholar]
  47. Steiner, K. & Malke, H. ( 2000; ). Life in protein-rich environments: the relA-independent response of Streptococcus pyogenes to amino acid starvation. Mol Microbiol 38, 1004–1016.
    [Google Scholar]
  48. Stevenson, L. H. ( 1977; ). A case for bacterial dormancy in aquatic systems. Microb Ecol 4, 127–133.[CrossRef]
    [Google Scholar]
  49. Van der Hoeven, J. S. & Camp, P. J. ( 1991; ). Synergistic degradation of mucin by Streptococcus oralis and Streptococcus sanguis in mixed chemostat cultures. J Dent Res 70, 1041–1044.[CrossRef]
    [Google Scholar]
  50. Walsh, S., Lappin-Scott, H. M., Stockdale, H. & Herbert, B. N. ( 1995; ). An assessment of the metabolic activity of starved and vegetative bacteria using two redox dyes. J Microbiol Methods 24, 1–9.[CrossRef]
    [Google Scholar]
  51. Watson, S. P., Clements, M. O. & Foster, S. J. ( 1998; ). Characterization of the starvation-survival response of Staphylococcus aureus. J Bacteriol 180, 1750–1758.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/016576-0
Loading
/content/journal/micro/10.1099/mic.0.2008/016576-0
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