1887

Abstract

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by strain PAO1 and its alginate-overproducing () and alginate-defective () variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg PAO and Alg PAO, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing () and Community Statistics () software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

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2004-07-01
2019-11-17
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References

  1. Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M. & Molin, S. ( 1998;). New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64, 2240–2246.
    [Google Scholar]
  2. Ashby, M. J., Neale, J. E., Knott, S. J. & Critchley, I. A. ( 1994;). Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. J Antimicrob Chemother 33, 443–452.[CrossRef]
    [Google Scholar]
  3. Baltimore, R. S. & Mitchell, M. ( 1980;). Immunologic investigations of mucoid strains of Pseudomonas aeruginosa: comparison of susceptibility to opsonic antibody in mucoid and nonmucoid strains. J Infect Dis 141, 238–247.[CrossRef]
    [Google Scholar]
  4. Baltimore, R. S., Christie, C. D. & Smith, G. J. ( 1989;). Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis.Implications for the pathogenesis of progressive lung deterioration. Am Rev Respir Dis 140, 1650–1661.[CrossRef]
    [Google Scholar]
  5. Boucher, J. C., Yu, H., Mudd, M. H. & Deretic, V. ( 1997;). Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection. Infect Immun 65, 3838–3846.
    [Google Scholar]
  6. Brown, M. R. W. & Williams, P. ( 1985;). The influence of environment on envelope properties affecting survival of bacteria in infections. Annu Rev Microbiol 39, 527–556.[CrossRef]
    [Google Scholar]
  7. Brown, M. R., Allison, D. G. & Gilbert, P. ( 1988;). Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother 22, 777–780.[CrossRef]
    [Google Scholar]
  8. Christensen, B. B., Sternberg, C., Andersen, J. B., Palmer, R. J., Jr, Nielsen, A. T., Givskov, M. & Molin, S. ( 1999;). Molecular tools for study of biofilm physiology. Methods Enzymol 310, 20–42.
    [Google Scholar]
  9. Cochran, W. L., Suh, S. J., McFeters, G. A. & Stewart, P. S. ( 2000;). Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine. J Appl Microbiol 88, 546–553.[CrossRef]
    [Google Scholar]
  10. Cormack, B. P., Valdivia, R. H. & Falkow, S. ( 1996;). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef]
    [Google Scholar]
  11. Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M. & Marrie, T. J. ( 1987;). Bacterial biofilms in nature and disease. Annu Rev Microbiol 41, 435–464.[CrossRef]
    [Google Scholar]
  12. Costerton, J. W., Lewandowski, Z., DeBeer, D., Caldwell, D., Korber, D. & James, G. ( 1994;). Biofilms, the customized microniche. J Bacteriol 176, 2137–2142.
    [Google Scholar]
  13. Davies, D. G. & Geesey, G. G. ( 1995;). Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl Environ Microbiol 61, 860–867.
    [Google Scholar]
  14. Davies, D. G., Chakrabarty, A. M. & Geesey, G. G. ( 1993;). Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol 59, 1181–1186.
    [Google Scholar]
  15. Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. ( 1998;). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295–298.[CrossRef]
    [Google Scholar]
  16. de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. ( 1990;). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568–6572.
    [Google Scholar]
  17. Deretic, V., Gill, J. F. & Chakrabarty, A. M. ( 1987;). Pseudomonas aeruginosa infection in cystic fibrosis: nucleotide sequence and transcriptional regulation of the algD gene. Nucleic Acids Res 11, 4567–4581.
    [Google Scholar]
  18. DeVries, C. A. & Ohman, D. E. ( 1994;). Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternative sigma factor, and shows evidence for autoregulation. J Bacteriol 176, 6677–6687.
    [Google Scholar]
  19. Doggett, R. G., Harrison, G. M., Stillwell, R. N. & Wallis, E. S. ( 1966;). An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. J Pediatr 68, 215–221.[CrossRef]
    [Google Scholar]
  20. Doig, P., Smith, N. R., Todd, T. & Irvin, R. T. ( 1987;). Characterization of the binding of Pseudomonas aeruginosa alginate to human epithelial cells. Infect Immun 55, 1517–1522.
    [Google Scholar]
  21. Evans, L. R. & Linker, A. ( 1973;). Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol 116, 915–924.
    [Google Scholar]
  22. Evans, D. J., Allison, D. G., Brown, M. R. & Gilbert, P. ( 1991;). Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother 27, 177–184.[CrossRef]
    [Google Scholar]
  23. Fick, R. B., Jr, Sonoda, F. & Hornick, D. B. ( 1992;). Emergence and persistence of Pseudomonas aeruginosa in the cystic fibrosis airway. Semin Respir Infect 7, 168–178.
    [Google Scholar]
  24. Gander, S. ( 1996;). Bacterial biofilms: resistance to antimicrobial agents. J Antimicrob Chemother 37, 1047–1050.[CrossRef]
    [Google Scholar]
  25. Garrett, E. S., Perlegas, D. & Wozniak, D. J. ( 1999;). Negative control of flagellum synthesis in Pseudomonas aeruginosa is modulated by the alternative sigma factor AlgT (AlgU). J Bacteriol 181, 7401–7404.
    [Google Scholar]
  26. Gilbert, P. & Brown, M. R. ( 1998;). Biofilms and β-lactam activity. J Antimicrob Chemother 41, 571–572.[CrossRef]
    [Google Scholar]
  27. Goldberg, J. B. & Ohman, D. E. ( 1984;). Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol 158, 1115–1121.
    [Google Scholar]
  28. Goldberg, J. B., Hatano, K. & Pier, G. B. ( 1993;). Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase. J Bacteriol 175, 1605–1611.
    [Google Scholar]
  29. Govan, J. R. & Harris, G. S. ( 1986;). Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. Microbiol Sci 3, 302–308.
    [Google Scholar]
  30. Hassett, D. J., Elkins, J. G., Ma, J. F. & McDermott, T. R. ( 1999;). Pseudomonas aeruginosa biofilm sensitivity to biocides: use of hydrogen peroxide as model antimicrobial agent for examining resistance mechanisms. Methods Enzymol 310, 599–608.
    [Google Scholar]
  31. Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F. & Haas, D. ( 2000;). Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol Plant Microbe Interact 13, 232–237.[CrossRef]
    [Google Scholar]
  32. Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M. & Parsek, M. R. ( 2001;). Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J Bacteriol 183, 5395–5401.[CrossRef]
    [Google Scholar]
  33. Hentzer, M., Riedel, K., Rasmussen, T. B. & 9 other authors ( 2002;). Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87–102.
    [Google Scholar]
  34. Herrero, M., de Lorenzo, V. & Timmis, K. N. ( 1990;). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172, 6557–6567.
    [Google Scholar]
  35. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. & Molin, S. ( 2000;). Quantification of biofilm structures by the novel computer program comstat. Microbiology 146, 2395–2407.
    [Google Scholar]
  36. Heydorn, A., Ersboll, B., Kato, J., Hentzer, M., Parsek, M. R., Tolker-Nielsen, T., Givskov, M. & Molin, S. ( 2002;). Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression. Appl Environ Microbiol 68, 2008–2017.[CrossRef]
    [Google Scholar]
  37. Høiby, N. ( 1974;). Pseudomonas aeruginosa infection in cystic fibrosis.Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response. Acta Pathol Microbiol Scand B 82, 551–558.
    [Google Scholar]
  38. Høiby, N. ( 1975;). Prevalence of mucoid strains of Pseudomonas aeruginosa in bacteriological specimens from patients with cystic fibrosis and patients with other diseases. Acta Pathol Microbiol Scand Suppl 83, 549–552.
    [Google Scholar]
  39. Holloway, B. W. & Morgan, A. F. ( 1986;). Genome organization in Pseudomonas. Annu Rev Microbiol 40, 79–105.[CrossRef]
    [Google Scholar]
  40. Hoyle, B. D. & Costerton, J. W. ( 1991;). Bacterial resistance to antibiotics: the role of biofilms. Prog Drug Res 37, 91–105.
    [Google Scholar]
  41. Hoyle, B. D., Williams, L. J. & Costerton, J. W. ( 1993;). Production of mucoid exopolysaccharide during development of Pseudomonas aeruginosa biofilms. Infect Immun 61, 777–780.
    [Google Scholar]
  42. Jensen, E. T., Kharazmi, A., Lam, K., Costerton, J. W. & Høiby, N. ( 1990;). Human polymorphonuclear leukocyte response to Pseudomonas aeruginosa grown in biofilms. Infect Immun 58, 2383–2385.
    [Google Scholar]
  43. Ji, Z. ( 2000;). Quantitative analysis of biofilm images using fractal dimensions. MSc thesis, University of Memphis, TN, USA.
  44. Kessler, B., de Lorenzo, V. & Timmis, K. N. ( 1992;). A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233, 293–301.[CrossRef]
    [Google Scholar]
  45. Kocharova, N. A., Hatano, K., Shaskov, A. S., Knirel, Y. A., Kochetkov, N. K. & Pier, G. B. ( 1989;). The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3. J Biol Chem 264, 15569–15573.
    [Google Scholar]
  46. Lam, J., Chan, R., Lam, K. & Costerton, J. W. ( 1980;). Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immun 28, 546–556.
    [Google Scholar]
  47. Lawrence, J. R., Korber, D. R., Hoyle, B. D., Costerton, J. W. & Caldwell, D. E. ( 1991;). Optical sectioning of microbial biofilms. J Bacteriol 173, 6558–6567.
    [Google Scholar]
  48. Maniatis, T., Fritsch, E. F. & Sambrook, J. ( 1982;). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  49. Marcus, H. & Baker, N. R. ( 1985;). Quantitation of adherence of mucoid and nonmucoid Pseudomonas aeruginosa to hamster tracheal epithelium. Infect Immun 47, 723–729.
    [Google Scholar]
  50. Mathee, K., McPherson, C. J. & Ohman, D. E. ( 1997;). Posttranslational control of the algT (algU)-encoded σ22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN). J Bacteriol 179, 3711–3720.
    [Google Scholar]
  51. Mathee, K., Ciofu, O., Givskov, M., Ohman, D. E., Molin, S., Høiby, N. & Kharazmi, A. ( 1999a;). Induction of Pseudomonas aeruginosa alginate production in vivo mediated by inflammatory response in lungs of cystic fibrosis patients. Clin Microbiol 5, S8–S9.[CrossRef]
    [Google Scholar]
  52. Mathee, K., Ciofu, O., Sternberg, C. & 9 other authors ( 1999b;). Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145, 1349–1357.[CrossRef]
    [Google Scholar]
  53. Mathee, K., Kharazmi, A. & Høiby, N. ( 2002;). Role of exopolysaccharide in biofilm matrix formation, the alginate paradigm. In Molecular Ecology of Biofilms, chapter 2. Edited by R. J. C. McLean & A. W. Decho. Wymondham, UK: Horizon Scientific Press.
  54. Narasimhan, G. ( 2004). Biofilm Image Processing Program at http://www.cs.fiu.edu/∼giri/BIP/
  55. Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J. ( 2001;). Role of alginate and its O-acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol 183, 1047–1057.[CrossRef]
    [Google Scholar]
  56. Olvera, C., Goldberg, J. B., Sanchez, R. & Soberon-Chavez, G. ( 1999;). The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis. FEMS Microbiol Lett 179, 85–90.[CrossRef]
    [Google Scholar]
  57. O'Toole, G. A. & Kolter, R. ( 1998;). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295–304.[CrossRef]
    [Google Scholar]
  58. Pedersen, S. S., Kharazmi, A., Espersen, F. & Høiby, N. ( 1990;). Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response. Infect Immun 58, 3363–3368.
    [Google Scholar]
  59. Pedersen, S. S., Høiby, N., Espersen, F. & Koch, C. ( 1992;). Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. Thorax 47, 6–13.[CrossRef]
    [Google Scholar]
  60. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. ( 1992;). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233.[CrossRef]
    [Google Scholar]
  61. Ramphal, R. & Pier, G. B. ( 1985;). Role of Pseudomonas aeruginosa mucoid exopolysaccharide in adherence to tracheal cells. Infect Immun 47, 1–4.
    [Google Scholar]
  62. Roychoudhury, S., May, T. B., Gill, J. F., Singh, S. K., Feingold, D. S. & Chakrabarty, A. M. ( 1989;). Purification and characterization of guanosine diphospho-d-mannose dehydrogenase.A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa. J Biol Chem 264, 9380–9385.
    [Google Scholar]
  63. Sauer, K. & Camper, A. K. ( 2001;). Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth. J Bacteriol 183, 6579–6589.[CrossRef]
    [Google Scholar]
  64. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies, D. G. ( 2002;). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184, 1140–1154.[CrossRef]
    [Google Scholar]
  65. Schierholz, J. M., Beuth, J., Konig, D., Nurnberger, A. & Pulverer, G. ( 1999;). Antimicrobial substances and effects on sessile bacteria. Zentralbl Bakteriol 289, 165–177[CrossRef]
    [Google Scholar]
  66. Song, Z., Wu, H., Ciofu, O., Kong, K.-F., Høiby, N., Rygaard, J., Kharazmi, A. & Mathee, K. ( 2003;). Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection. J Med Microbiol 52, 731–740.[CrossRef]
    [Google Scholar]
  67. Stoodley, P., Dodds, I., Boyle, J. D. & Lappin-Scott, H. M. ( 1999;). Influence of hydrodynamics and nutrients on biofilm structure. J Appl Microbiol 85, S19–S28.
    [Google Scholar]
  68. Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J. D., Lappin-Scott, H. M. & Costerton, J. W. ( 2001;). Growth and detachment of cell clusters from mature mixed-species biofilms. Appl Environ Microbiol 67, 5608–5613.[CrossRef]
    [Google Scholar]
  69. Wolfaardt, G. M., Lawrence, J. R., Robarts, R. D., Caldwell, S. J. & Caldwell, D. E. ( 1994;). Multicellular organization in a degradative biofilm community. Appl Environ Microbiol 60, 434–446.
    [Google Scholar]
  70. Wozniak, D. J., Wyckoff, T. J., Starkey, M., Keyser, R., Azadi, P., O'Toole, G. A. & Parsek, M. R. ( 2003;). Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci U S A 100, 7907–7912.[CrossRef]
    [Google Scholar]
  71. Yang, X., Beyenal, H., Harkin, G. & Lewandowski Z. ( 2000;). Quantifying biofilm structure using image analysis. J Microbiol Methods 39, 109–119.[CrossRef]
    [Google Scholar]
  72. Yu, H., Hanes, M., Chrisp, C. E., Boucher, J. C. & Deretic, V. ( 1998;). Microbial pathogenesis in cystic fibrosis: pulmonary clearance of mucoid Pseudomonas aeruginosa and inflammation in a mouse model of repeated respiratory challenge. Infect Immun 66, 280–288.
    [Google Scholar]
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