Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Physiological heterogeneity in biofilms

Nature Reviews Microbiology volume 6, pages 199–210 (2008)Cite this article

Key Points

  • Bacterial cells growing in biofilms are physiologically distinct from free-swimming planktonic cells. For example, differences have been shown in motility, polysaccharide production, antibiotic tolerance and global proteomic and transcriptomic profiles.

  • Bacterial cells within biofilms can also be physiologically distinct from adjacent cells on a micrometre scale.

  • Chemical heterogeneities are established in biofilms primarily owing to bacterial metabolic activity and solute diffusion. Chemical gradients of oxygen, nutrients, bacterial waste products and bacterial signalling compounds can therefore be established, thereby generating unique environmental conditions for the cells.

  • Bacterial adaptation to chemical gradients in biofilms includes differences in gene expression and protein production. Also relevant are mutation and selection for the fittest organisms in a particular microenvironment, as well as the response that is due to stochastic gene expression.

  • Techniques for characterizing gene expression and physiological activities have been applied to bacterial biofilms to characterize the local activity of cells within biofilms. Examples that used these techniques are discussed, including the advantages and disadvantages of using particular techniques to characterize subsets of cells from within biofilms.

Abstract

Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Biofilm heterogeneity.
Figure 2: Chemical heterogeneity in biofilms.
Figure 3: Physiological heterogeneity in a single-species biofilm.
Figure 4: Physiological heterogeneity in a mixed-species biofilm.
Figure 5: Multiplicity of phenotypic states in biofilms.

Similar content being viewed by others

References

  1. Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. Microbiol. 2, 95–108 (2004).

    Article  CAS  Google Scholar 

  2. Drenkard, E. & Ausubel, F. M. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Mah, T. F. et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426, 306–310 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Bjarnsholt, T. et al. Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151, 373–383 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Jesaitis, A. J. et al. Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J. Immunol. 171, 4329–4339 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Bryers, J. D. in Biofilms (eds Characklis, W. G. & Marshall, K. C.) 733–773 (Wiley-Intersciences, New York, 1990).

    Google Scholar 

  7. Bryers, J. D. & Characklis, W. G. in Biofilms (eds Characklis, W. G. & Marshall, K. C.) 671–696 (Wiley-Intersciences, New York, 1990).

    Google Scholar 

  8. Belas, R., Simon, M. & Silverman, M. Regulation of lateral flagella gene transcription in Vibrio parahaemolyticus. J. Bacteriol. 167, 210–218 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. McCarter, L. & Silverman, M. Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus. Mol. Microbiol. 4, 1057–1062 (1990).

    Article  CAS  PubMed  Google Scholar 

  10. Davies, D. G. & Geesey, G. G. Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl. Environ. Microbiol. 61, 860–867 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Dalton, H. M. et al. Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure. J. Bacteriol. 176, 6900–6906 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. An, D. & Parsek, M. R. The promise and peril of transcriptional profiling in biofilm communities. Curr. Opin. Microbiol. 10, 292–296 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies, D. G. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184, 1140–1154 (2002). Demonstrated that P. aeruginosa undergoes a range of physiological changes during the initiation, development, maturation and dispersal of biofilms, and that each physiological state is accompanied by dramatic changes in the proteome profiles of the cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Southey-Pillig, C. J., Davies, D. G. & Sauer, K. Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J. Bacteriol. 187, 8114–8126 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. O'Toole, G. A. & Kolter, R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28, 449–461 (1998). Describes a microtitre dish screening assay that was used to identify transposon mutants that are unable to form biofilms, thereby linking distinct genetic loci to biofilm formation.

    Article  CAS  PubMed  Google Scholar 

  16. Patrauchan, M. A., Sarkisova, S. & Franklin, M. J. Strain specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium. Microbiology 153, 3838–3851 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Glud, R. N., Ramsing, N. B. & Revsbech, N. P. Photosynthesis and photosynthesis-coupled respiration in natural biofilms quantified with oxygen microsensors. J. Phycol. 28, 51–60 (1992).

    Article  Google Scholar 

  18. Ramsing, N. B., Kuhl, M. & Jorgensen, B. B. Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes. Appl. Environ. Microbiol. 59, 3840–3849 (1993). Reports microelectrode measurements of concentration gradients in biofilms, and integrates these with ecological analysis by the use of FISH probes.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. De Beer, D., Stoodley, P., Roe, F. & Lewandowski, Z. Effects of biofilm structure on oxygen distribution and mass transport. Biotechnol. Bioeng. 43, 1131–1138 (2004).

    Article  Google Scholar 

  20. Zhang, T., Fu, Y. & Bishop, P. Competition for substrate and space in biofilms. Water Environ. Res. 67, 992–1003 (1995).

    Article  CAS  Google Scholar 

  21. Schramm, A. et al. Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes. Appl. Environ. Microbiol. 62, 4641–4647 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T. & McFeters, G. A. Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl. Environ. Microbiol. 64, 4035–4039 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Okabe, S., Satoh, H. & Watanabe, Y. In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes. Appl. Environ. Microbiol. 65, 3182–3191 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Rani, S. A. et al. Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal diverse physiological states. J. Bacteriol. 189, 4223–4233 (2007). Experimental demonstration of spatial heterogeneity in oxygen concentration and anabolic protein and DNA synthesis.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Allewalt, J. P., Bateson, M. M., Revsbech, N. P., Slack, K. & Ward, D. M. Effect of temperature and light on growth of and photosynthesis by Synechococcus isolates typical of those predominating in the octopus spring microbial mat community of Yellowstone National Park. Appl. Environ. Microbiol. 72, 544–550 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ward, D. M. Microbial diversity in natural environments: focusing on fundamental questions. Antonie Van Leeuwenhoek 90, 309–324 (2006).

    Article  PubMed  Google Scholar 

  27. Kolenbrander, P. E. Oral microbial communities: biofilms, interactions, and genetic systems. Annu. Rev. Microbiol. 54, 413–437 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Stewart, P. S. Diffusion in biofilms. J. Bacteriol. 185, 1485–1491 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Damgaard, L. R., Nielsen, L. P. & Revsbech, N. P. Methane microprofiles in a sewage biofilm determined with a microscale biosensor. Water Res. 35, 1379–1386 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. De Beer, D., Schramm, A., Santegoeds, C. M. & Kuhl, M. A nitrite microsensor for profiling environmental biofilms. Appl. Environ. Microbiol. 63, 973–977 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kuhl, M., Steuckart, C., Eickert, G. & Jeroschewski, P. A H2S microsensor for profiling biofilms and sediments: application in an acidic lake sediment. Aquat. Microb. Ecol. 15, 201–209 (1998).

    Article  Google Scholar 

  32. Okabe, S., Itoh, T., Satoh, H. & Watanabe, Y. Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms. Appl. Environ. Microbiol. 65, 5107–5116 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. De Beer, D., Glud, A., Epping, E. & Kuhl, M. A fast-responding CO2 microelectrode for profiling sediments, microbial mats, and biofilms. Limnol. Oceanogr. 42, 1590–1600 (1997).

    Article  CAS  Google Scholar 

  34. Beyenal, H., Davis, C. & Lewandowski, Z. An improved Severinghaus-type carbon dioxide microelectrode for use in biofilms. Sens Actuators B 97, 202–210 (2004).

    Article  CAS  Google Scholar 

  35. Liu, X., Roe, F., Jesaitis, A. J. & Lewandowski, Z. Resistance of biofilms to the catalase inhibitor 3-amino-1,2,4-triazole. Biotechnol. Bioeng. 59, 156–162 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. De Beer, D., Srinivasan, R. & Stewart, P. S. Direct measurement of chlorine penetration into biofilms during disinfection. Appl. Environ. Microbiol. 60, 4339–4344 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jang, A., Szabo, J., Hosni, A. A., Coughlin, M. & Bishop, P. L. Measurement of chlorine dioxide penetration in dairy process pipe biofilms during disinfection. Appl. Microbiol. Biotechnol. 72, 368–376 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. De Beer, D., Huisman, J., Van den Heuvel, J. & Ottengraf, S. The effect of pH profiles in methanogenic aggregates on the kinetics of acetate conversion. Water Res. 26, 1329–1336 (1992).

    Article  CAS  Google Scholar 

  39. Huang, C. T., Yu, F. P., McFeters, G. & Stewart, P. S. Nonuniform spatial patterns of respiratory activity within biofilms during disinfection. Appl. Environ. Microbiol. 61, 2252–2256 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Wentland, E., Stewart, P. S., Huang, C. T. & McFeters, G. A. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol. Prog. 12, 316–321 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Huang, C. T., Xu, K. D., McFeters, G. A. & Stewart, P. S. Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation. Appl. Environ. Microbiol. 64, 1526–1531 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kuhl, M. & Jorgensen, B. B. Microsensor measurements of sulfate reduction and sulfide oxidation in compact microbial communities of aerobic biofilms. Appl. Environ. Microbiol. 58, 1164–1174 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Otto, K. & Silhavy, T. J. Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc. Natl Acad. Sci. USA 99, 2287–2292 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kolenbrander, P. E. et al. Communication among oral bacteria. Microbiol. Mol. Biol. Rev. 66, 486–505 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964 (2000).

    Article  CAS  PubMed  Google Scholar 

  46. Allegrucci, M. & Sauer, K. Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms. J. Bacteriol. 189, 2030–2038 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Hansen, S. K., Rainey, P. B., Haagensen, J. A. & Molin, S. Evolution of species interactions in a biofilm community. Nature 445, 533–536 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Valle, J., Vergara-Irigaray, M., Merino, N., Penades, J. R. & Lasa, I. σB regulates IS256-mediated Staphylococcus aureus biofilm phenotypic variation. J. Bacteriol. 189, 2886–2896 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yildiz, F. H. & Schoolnik, G. K. Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl Acad. Sci. USA 96, 4028–4033 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. McEllistrem, M. C., Ransford, J. V. & Khan, S. A. Characterization of in vitro biofilm-associated pneumococcal phase variants of a clinically relevant serotype 3 clone. J. Clin. Microbiol. 45, 97–101 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Koh, K. S. et al. Phenotypic diversification and adaptation of Serratia marcescens MG1 biofilm-derived morphotypes. J. Bacteriol. 189, 119–130 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Kirisits, M. J., Prost, L., Starkey, M. & Parsek, M. R. Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 71, 4809–4821 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Haussler, S. Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa. Environ. Microbiol. 6, 546–551 (2004).

    Article  PubMed  Google Scholar 

  54. Nguyen, D. & Singh, P. K. Evolving stealth: genetic adaptation of Pseudomonas aeruginosa during cystic fibrosis infections. Proc. Natl Acad. Sci. USA 103, 8305–8306 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hansen, S. K. et al. Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6. J. Bacteriol. 189, 4932–4943 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Boles, B. R., Thoendel, M. & Singh, P. K. Self-generated diversity produces “insurance effects” in biofilm communities. Proc. Natl Acad. Sci. USA 101, 16630–16635 (2004). Demonstrated that P. aeruginosa undergoes genetic diversification during short-term biofilm growth, which might allow increased resistance of the population to environmental stresses.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cooper, T. F., Beaumont, H. J. & Rainey, P. B. Biofilm diversity as a test of the insurance hypothesis. Microbiology 151, 2815–2816; discussion 2816–2818 (2005).

    Article  CAS  PubMed  Google Scholar 

  58. Deziel, E., Comeau, Y. & Villemur, R. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J. Bacteriol. 183, 1195–1204 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Baty, A. M. et al. Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation. Appl. Environ. Microbiol. 66, 3566–3573 (2000). Used a gfp reporter to the chitinase promoter to demonstrate that biofilms of the marine bacterium Pseudoalteromonas sp. resulted in two distinct subpopulations with respect to chitinase activity; this seems to have been due to stochastic gene expression.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Baty, A. M., Eastburn, C. C., Techkarnjanaruk, S., Goodman, A. E. & Geesey, G. G. Spatial and temporal variations in chitinolytic gene expression and bacterial biomass production during chitin degradation. Appl. Environ. Microbiol. 66, 3574–3585 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Hasty, J., Pradines, J., Dolnik, M. & Collins, J. J. Noise-based switches and amplifiers for gene expression. Proc. Natl Acad. Sci. USA 97, 2075–2080 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. McAdams, H. H. & Arkin, A. Stochastic mechanisms in gene expression. Proc. Natl Acad. Sci. USA 94, 814–819 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    Article  CAS  PubMed  Google Scholar 

  64. Chai, Y., Chu, F., Kolter, R. & Losick, R. Bistability and biofilm formation in Bacillus subtilis. Mol. Microbiol. 67, 254–263 (2007). Demonstrated that matrix polymer production in B. subtilis is under the control of a bistable mechanism that involves the repressor SinR and its anti-repressor SinI.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Branda, S. S., Chu, F., Kearns, D. B., Losick, R. & Kolter, R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59, 1229–1238 (2006).

    Article  CAS  PubMed  Google Scholar 

  66. Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R. & Kolter, R. Fruiting body formation by Bacillus subtilis. Proc. Natl Acad. Sci. USA 98, 11621–11626 (2001). Showed that, when cultured in biofilms, B. subtilis spore formation is spatially organized and occurs in structures that resemble fruiting bodies.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Keren, I., Kaldalu, N., Spoering, A., Wang, Y. & Lewis, K. Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230, 13–18 (2004).

    Article  CAS  PubMed  Google Scholar 

  68. Lewis, K. Persister cells, dormancy and infectious disease. Nature Rev. Microbiol. 5, 48–56 (2007).

    Article  CAS  Google Scholar 

  69. Levin, B. R. & Rozen, D. E. Non-inherited antibiotic resistance. Nature Rev. Microbiol. 4, 556–562 (2006).

    Article  CAS  Google Scholar 

  70. Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004). A microfluidics approach was used to investigate the pre-existing heterogeneity in growth rates of bacteria at the single-cell level. Cells that had reduced growth rates were linked with persistence to antibiotic challenge.

    Article  CAS  PubMed  Google Scholar 

  71. Kussell, E., Kishony, R., Balaban, N. Q. & Leibler, S. Bacterial persistence: a model of survival in changing environments. Genetics 169, 1807–1814 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Correia, F. F. et al. Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J. Bacteriol. 188, 8360–8367 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Korch, S. B. & Hill, T. M. Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation. J. Bacteriol. 188, 3826–3836 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Li, Y. & Zhang, Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob. Agents Chemother. 51, 2092–2099 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Spoering, A. L., Vulic, M. & Lewis, K. GlpD and PlsB participate in persister cell formation in Escherichia coli. J. Bacteriol. 188, 5136–5144 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Rodriguez, G. G., Phipps, D., Ishiguro, K. & Ridgway, H. F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58, 1801–1808 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Schaule, G., Flemming, H. C. & Ridgway, H. F. Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl. Environ. Microbiol. 59, 3850–3857 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Zheng, Z. & Stewart, P. S. Growth limitation of Staphylococcus epidermidis in biofilms contributes to rifampin tolerance. Biofilms 1, 31–45 (2004).

    Article  Google Scholar 

  79. Nielsen, J. L., Aquino de Muro, M. & Nielsen, P. H. 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 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Ullrich, S., Karrasch, B., Hoppe, H., Jeskulke, K. & Mehrens, M. Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl. Environ. Microbiol. 62, 4587–4593 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Smith, J. J. & McFeters, G. A. Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli. J. Appl. Bacteriol. 80, 209–215 (1996).

    Article  CAS  PubMed  Google Scholar 

  82. Korber, D. R., Choi, A., Wolfaardt, G. M., Ingham, S. C. & Caldwell, D. E. Substratum topography influences susceptibility of Salmonella enteritidis biofilms to trisodium phosphate. Appl. Environ. Microbiol. 63, 3352–3358 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Lindsay, D., Brozel, V. S., Mostert, J. F. & von Holy, A. Differential efficacy of a chlorine dioxide-containing sanitizer against single species and binary biofilms of a dairy-associated Bacillus cereus and a Pseudomonas fluorescens isolate. J. Appl. Microbiol. 92, 352–361 (2002).

    Article  CAS  PubMed  Google Scholar 

  84. Hentzer, M. et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 22, 3803–3815 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Teitzel, G. M. & Parsek, M. R. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 69, 2313–2320 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Hope, C. K. & Wilson, M. Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob. Agents Chemother. 48, 1461–1468 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Barraud, N. et al. Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J. Bacteriol. 188, 7344–7353 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Berney, M., Hammes, F., Bosshard, F., Weilenmann, H. U. & Egli, T. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl. Environ. Microbiol. 73, 3283–3290 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Biggerstaff, J. P. et al. New methodology for viability testing in environmental samples. Mol. Cell. Probes 20, 141–146 (2006).

    Article  CAS  PubMed  Google Scholar 

  90. Stocks, S. M. Mechanism and use of the commercially available viability stain, BacLight. Cytometry A 61, 189–195 (2004).

    Article  CAS  PubMed  Google Scholar 

  91. Suci, P. A. & Tyler, B. J. Action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage Candida albicans biofilm. Antimicrob. Agents Chemother. 46, 3522–3531 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Banin, E., Brady, K. M. & Greenberg, E. P. Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm. Appl. Environ. Microbiol. 72, 2064–2069 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Haagensen, J. A. et al. Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms. J. Bacteriol. 189, 28–37 (2007).

    Article  CAS  PubMed  Google Scholar 

  94. Kaneko, Y., Thoendel, M., Olakanmi, O., Britigan, B. E. & Singh, P. K. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J. Clin. Invest. 117, 877–888 (2007). Together with reference 93, demonstrated the differing antimicrobial susceptibility of spatially segregated populations within a biofilm.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Auschill, T. M. et al. Spatial distribution of vital and dead microorganisms in dental biofilms. Arch. Oral Biol. 46, 471–476 (2001).

    Article  CAS  PubMed  Google Scholar 

  96. Honraet, K., Goetghebeur, E. & Nelis, H. J. Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms. J. Microbiol. Methods 63, 287–295 (2005).

    Article  CAS  PubMed  Google Scholar 

  97. Hamasaki, K., Taniguchi, A., Tada, Y., Long, R. A. & Azam, F. Actively growing bacteria in the inland sea of Japan, identified by combined bromodeoxyuridine immunocapture and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 73, 2787–2798 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Hjort, K., Lembke, A., Speksnijder, A., Smalla, K. & Jansson, J. K. Community structure of actively growing bacterial populations in plant pathogen suppressive soil. Microb. Ecol. 53, 399–413 (2007).

    Article  PubMed  Google Scholar 

  99. Jackson, K. D., Starkey, M., Kremer, S., Parsek, M. R. & Wozniak, D. J. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J. Bacteriol. 186, 4466–4475 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Friedman, L. & Kolter, R. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J. Bacteriol. 186, 4457–4465 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 183, 1047–1057 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C. & Mattick, J. S. Extracellular DNA required for bacterial biofilm formation. Science 295, 1487 (2002).

    Article  CAS  PubMed  Google Scholar 

  103. Allesen-Holm, M. et al. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Microbiol. 59, 1114–1128 (2006).

    Article  CAS  PubMed  Google Scholar 

  104. Sarkisova, S., Patrauchan, M. A., Berglund, D., Nivens, D. E. & Franklin, M. J. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J. Bacteriol. 187, 4327–4337 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Neu, T., Swerhone, G. D. & Lawrence, J. R. Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147, 299–313 (2001).

    Article  CAS  PubMed  Google Scholar 

  106. Lawrence, J. R., Swerhone, G. D., Kuhlicke, U. & Neu, T. R. In situ evidence for microdomains in the polymer matrix of bacterial microcolonies. Can. J. Microbiol. 53, 450–458 (2007).

    Article  CAS  PubMed  Google Scholar 

  107. Laue, H. et al. Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae. Microbiology 152, 2909–2918 (2006).

    Article  CAS  PubMed  Google Scholar 

  108. Strathmann, M., Wingender, J. & Flemming, H. C. Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 50, 237–248 (2002).

    Article  CAS  PubMed  Google Scholar 

  109. Sternberg, C. et al. Distribution of bacterial growth activity in flow-chamber biofilms. Appl. Environ. Microbiol. 65, 4108–4117 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Werner, E. et al. Stratified growth in Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 70, 6188–6196 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Teal, T. K., Lies, D. P., Wold, B. J. & Newman, D. K. Spatiometabolic stratification of Shewanella oneidensis biofilms. Appl. Environ. Microbiol. 72, 7324–7330 (2006). Used the unstable gfp reporter gene in S. oneidensis to demonstrate the reproducible stratification of metabolic programming to local environmental conditions and biofilm developmental processes.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Walters, M. C., Roe, F., Bugnicourt, A., Franklin, M. J. & Stewart, P. S. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob. Agents Chemother. 47, 317–323 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Borriello, G., Richards, L., Ehrlich, G. D. & Stewart, P. S. Arginine or nitrate enhances antibiotic susceptibility of Pseudomonas aeruginosa in biofilms. Antimicrob. Agents Chemother. 50, 382–384 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Bagge, N. et al. Dynamics and spatial distribution of β-lactamase expression in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 48, 1168–1174 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. De Kievit, T. R. & Iglewski, B. H. Quorum sensing, gene expression, and Pseudomonas biofilms. Methods Enzymol. 310, 117–128 (1999).

    Article  CAS  PubMed  Google Scholar 

  116. Lequette, Y. & Greenberg, E. P. Timing and localization of rhamnolipid synthesis gene expression in Pseudomonas aeruginosa biofilms. J. Bacteriol. 187, 37–44 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Yang, L. et al. Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa. Microbiology 153, 1318–1328 (2007).

    Article  CAS  PubMed  Google Scholar 

  118. Yarwood, J. M., Bartels, D. J., Volper, E. M. & Greenberg, E. P. Quorum sensing in Staphylococcus aureus biofilms. J. Bacteriol. 186, 1838–1850 (2004). Used a reporter gene in Staphylococcus aureus to follow complex spatial and temporal patterns of expression of a quorum-sensing gene.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. De Kievit, T. R. et al. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 45, 1761–1770 (2001). Used reporter genes in P. aeruginosa to quantify and map spatial and temporal patterns of multidrug efflux-pump expression in biofilms.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Uppuluri, P., Sarmah, B. & Chaffin, W. L. Candida albicans SNO1 and SNZ1 expressed in stationary-phase planktonic yeast cells and base of biofilm. Microbiology 152, 2031–2038 (2006).

    Article  CAS  PubMed  Google Scholar 

  121. Moller, S. et al. In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members. Appl. Environ. Microbiol. 64, 721–732 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Klausen, M., Aaes-Jorgensen, A., Molin, S. & Tolker-Nielsen, T. Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol. Microbiol. 50, 61–68 (2003). Used fluorescent-protein-labelled wild-type and motility-mutant strains of P. aeruginosa to show the role of cell migration and type IV pili in biofilm developmental processes.

    Article  CAS  PubMed  Google Scholar 

  123. Amann, R. I. Fluorescently labelled, rRNA-targeted oligonucleotide probes in the study of microbial ecology. Mol. Ecol. 4, 543–554 (1995).

    Article  CAS  Google Scholar 

  124. DeLong, E. F., Wickham, G. S. & Pace, N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 1360–1363 (1989).

    Article  CAS  PubMed  Google Scholar 

  125. Moller, S., Pedersen, A. R., Poulsen, L. K., Arvin, E. & Molin, S. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy. Appl. Environ. Microbiol. 62, 4632–4640 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Xu, K. D., McFeters, G. A. & Stewart, P. S. Biofilm resistance to antimicrobial agents. Microbiology 146, 547–549 (2000).

    Article  CAS  PubMed  Google Scholar 

  127. Jang, A., Okabe, S., Watanabe, Y., Kim, I. & Bishop, P. Measurement of growth rate of ammonia oxidizing bacteria in partially submerged rotating biological contactor by fluorescent in situ hybridization (FISH). J. Environ. Sci. Eng. 4, 413–420 (2005).

    Article  CAS  Google Scholar 

  128. Lehtola, M. J., Torvinen, E., Miettinen, I. T. & Keevil, C. W. Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms. Appl. Environ. Microbiol. 72, 848–853 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Ferrari, B. C., Tujula, N., Stoner, K. & Kjelleberg, S. Catalyzed reporter deposition-fluorescence in situ hybridization allows for enrichment-independent detection of microcolony-forming soil bacteria. Appl. Environ. Microbiol. 72, 918–922 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Pernthaler, A., Pernthaler, J. & Amann, R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 3094–3101 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Fazi, S., Amalfitano, S., Pernthaler, J. & Puddu, A. Bacterial communities associated with benthic organic matter in headwater stream microhabitats. Environ. Microbiol. 7, 1633–1640 (2005).

    Article  CAS  PubMed  Google Scholar 

  132. Magnuson, T. S., Neal, A. L. & Geesey, G. G. Combining in situ reverse transcriptase polymerase chain reaction, optical microscopy, and X-ray photoelectron spectroscopy to investigate mineral surface-associated microbial activities. Microb. Ecol. 48, 578–588 (2004).

    Article  CAS  PubMed  Google Scholar 

  133. Wagner, M., Nielsen, P. H., Loy, A., Nielsen, J. L. & Daims, H. Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17, 83–91 (2006).

    Article  CAS  PubMed  Google Scholar 

  134. Huang, W. E. et al. Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ. Microbiol. 9, 1878–1889 (2007).

    Article  CAS  PubMed  Google Scholar 

  135. Lee, N. et al. Combination of fluorescent in situ hybridization and microautoradiography — a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289–1297 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Ouverney, C. C. & Fuhrman, J. A. Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746–1752 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Kindaichi, T., Ito, T. & Okabe, S. Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization. Appl. Environ. Microbiol. 70, 1641–1650 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Ito, T., Nielsen, J. L., Okabe, S., Watanabe, Y. & Nielsen, P. H. Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic–anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization. Appl. Environ. Microbiol. 68, 356–364 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Bonner, R. F. et al. Laser capture microdissection: molecular analysis of tissue. Science 278, 1481–1483 (1997).

    Article  CAS  PubMed  Google Scholar 

  140. Emmert-Buck, M. R. et al. Laser capture microdissection. Science 274, 998–1001 (1996).

    Article  CAS  PubMed  Google Scholar 

  141. Lenz, A. P., Williamson, K., Pitts, B., Stewart, P. S. & Franklin, M. J. in Abstr. Gen. Meet. Am. Soc. Microbiol. (American Society of Microbiology, Washington D.C., 2007).

  142. Rani, S. A., Pitts, B. & Stewart, P. S. Rapid diffusion of fluorescent tracers into Staphylococcus epidermidis biofilms visualized by time lapse microscopy. Antimicrob. Agents Chemother. 49, 728–732 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Stewart, P. S. et al. Observations of cell cluster hollowing in Staphylococcus epidermidis biofilms. Lett. Appl. Microbiol. 44, 454–457 (2007).

    Article  CAS  PubMed  Google Scholar 

  144. Seymour, J. D., Codd, S. L., Gjersing, E. L. & Stewart, P. S. Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor. J. Magn. Reson. 167, 322–327 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Work in the laboratory of P.S.S. has been supported by National Institutes of Health grant 5R01GM67245 and an award from the W. M. Keck Foundation. M.J.F.'s work on this topic is supported by Public Health Service grant AI-065906 from the National Institute of Allergy and Infectious Diseases.

Author information

Authors and Affiliations

  1. Center for Biofilm Engineering, Montana State University, Bozeman, 59717-3980, Montana, USA

    Philip S. Stewart & Michael J. Franklin

  2. Department of Chemical and Biological Engineering, Montana State University, Bozeman, 59717-3980, Montana, USA

    Philip S. Stewart

  3. Department of Microbiology, Montana State University, Bozeman, 59717-3980, Montana, USA

    Michael J. Franklin

Authors
  1. Philip S. Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael J. Franklin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philip S. Stewart.

Related links

Related links

DATABASES

Entrez Genome Project

Bacillus subtilis

Escherichia coli

Pseudomonas aeruginosa

Pseudomonas putida

Pseudomonas syringae

Shewanella oneidensis

Staphylococcus epidermidis

Streptococcus pneumoniae

Vibrio cholerae

FURTHER INFORMATION

Philip S. Stewart's homepage

Montana State University's Center for Biofilm Engineering Resource Library (biofilm movies)

Glossary

Extracellular polymeric substance

A polymer, such as a polysaccharide, protein or nucleic acid, that is secreted by bacteria and forms a hydrated gel-like slime. Extracellular polymeric substances hold the biofilm together, and might serve other functions, such as nutrient trapping and protection from antimicrobial challenges.

Reaction–diffusion theory.

A mathematical analysis of the distribution of a chemical solute in space and time that results from the interaction of two processes: reaction of the solute and its transport by diffusion. Reaction–diffusion interactions generate spatial gradients in the concentration of reacting solutes.

Persister cell

A metabolically quiescent cell that neither grows nor dies when exposed to cidal concentrations of antimicrobial compounds.

Peptide nucleic acid probe–FISH

Sequence-specific identification of nucleic acids using fluorescently labelled probes that contain peptide backbones. Useful for identifying individual cells in biofilms, generally by probing specific ribosomal RNA sequences.

Catalyzed reporter deposition–FISH

A method for increasing the sensitivity of FISH by using horseradish peroxidase-labelled oligonucleotide probes. The enzyme catalyzes the deposition of tyramine molecules, which results in fluorescent signal amplification at the site of probe hybridization.

In situ reverse-transcription polymerase chain reaction

A method to convert mRNA to cDNA and amplify the cDNA from cells that are fixed on microscope slides. Gene expression from individual cells is then assayed by using sequence-specific fluorescent probes that hybridize to the amplified product.

Microautoradiography

A method in which the activity of individual cells is determined by assaying incorporation of a radiolabelled substrate into cell material. Cell activity is determined by exposing labelled cells to photographic emulsion and quantifying exposed silver grains adjacent to the cells.

Laser capture microdissection microscopy

A microscopic method in which a laser is used to excise subsets of cells from the surrounding population. The excised cells can then be isolated for further analysis by laser catapulting.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stewart, P., Franklin, M. Physiological heterogeneity in biofilms. Nat Rev Microbiol 6, 199–210 (2008). https://doi.org/10.1038/nrmicro1838

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrmicro1838

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing