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:

Applying insights from biofilm biology to drug development — can a new approach be developed?

Nature Reviews Drug Discovery volume 12, pages 791–808 (2013)Cite this article

Subjects

Key Points

  • Little is known about the pathogenesis of infections caused by bacteria that grow as aggregates in biofilms.

  • Biofilm infections resist innate and adaptive immune defence mechanisms as well as antibiotics.

  • No drugs are available that specifically target bacteria in biofilms.

  • This Review presents current insights into biofilm physiology and pathology, and discusses strategies for the development of anti-biofilm drugs.

Abstract

Most of the research on bacterial pathogenesis has focused on acute infections, but much less is known about the pathogenesis of infections caused by bacteria that grow as aggregates in biofilms. These infections tend to be chronic as they resist innate and adaptive immune defence mechanisms as well as antibiotics, and the treatment of biofilm infections presents a considerable unmet clinical need. To date, there are no drugs that specifically target bacteria in biofilms; however, several approaches are in early-stage development. Here, we review current insights into biofilm physiology and pathology, and discuss how a deep insight into the physical and biological characteristics of biofilms can inform therapeutic strategies and molecular targets for the development of anti-biofilm drugs.

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: The development of bacterial biofilms from planktonic bacteria.
Figure 2: Pseudomonas aeruginosa biofilms visualized by confocal laser scanning microscopy.
Figure 3: Sensitivity of mechanically disrupted Pseudomonas aeruginosa biofilms to tobramycin.
Figure 4: The four anti-biofilm strategies.
Figure 5: The basic quorum sensor.
Figure 6: Garlic-dependent sensitivity of Pseudomonas aeruginosa biofilms towards polymorphonuclear leukocytes.
Figure 7: Pharmacokinetics and pharmacodynamics of antibiotics used to treat biofilm infections.

Similar content being viewed by others

References

  1. Murray, C. J. & Lopez, A. D. Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 349, 1498–1504 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. & Lappin-Scott, H. M. Microbial biofilms. Annu. Rev. Microbiol. 49, 711–745 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Potera, C. Studying slime. Environ. Health Perspect. 106, A604–A606 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Martinez, J. L. The role of natural environments in the evolution of resistance traits in pathogenic bacteria. Proc. Biol. Sci. 276, 2521–2530 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Høiby, N. Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey. Acta. Pathol. Microbiol. Scand. Suppl. 262, 1–96 (1977).

    Google Scholar 

  6. McCoy, W. F., Bryers, J. D., Robbins, J. & Costerton, J. W. Observations of fouling biofilm formation. Can. J. Microbiol. 27, 910–917 (1981).

    Article  CAS  PubMed  Google Scholar 

  7. Marrie, T. J. & Costerton, J. W. Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraarterial catheters. J. Clin. Microbiol. 19, 687–693 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Costerton, J. W. How bacteria stick — a citation classic commentary on how bacteria stick by Costerton, J. W., Geesey, G. G., and Cheng, K. J. Citation classics 48, 18 (1989).

    Google Scholar 

  9. Islam, M. S., Richards, J. P. & Ojha, A. K. Targeting drug tolerance in mycobacteria: a perspective from mycobacterial biofilms. Expert. Rev. Anti. Infect. Ther. 10, 1055–1066 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Muller, F. M., Seidler, M. & Beauvais, A. Aspergillus fumigatus biofilms in the clinical setting. Med. Mycol. 49 (Suppl. 1), 96–100 (2011).

    Article  Google Scholar 

  11. Cuellar-Cruz, M., Lopez-Romero, E., Villagomez-Castro, J. C. & Ruiz-Baca, E. Candida species: new insights into biofilm formation. Future. Microbiol. 7, 755–771 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Donlan, R. M. & Costerton, J. W. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15, 167–193 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Parsek, M. R. & Singh, P. K. Bacterial biofilms: an emerging link to disease pathogenesis. Annu. Rev. Microbiol. 57, 677–701 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. 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 

  15. Burmolle, M. et al. Biofilms in chronic infections — a matter of opportunity — monospecies biofilms in multispecies infections. FEMS Immunol. Med. Microbiol. 59, 324–336 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Jensen, P. O., Givskov, M., Bjarnsholt, T. & Moser, C. The immune system versus Pseudomonas aeruginosa biofilms. FEMS Immunol. Med. Microbiol. 59, 292–305 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Pamp, S. J., Gjermansen, M., Johansen, H. K. & Tolker-Nielsen, T. Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB–oprM genes. Mol. Microbiol. 68, 223–240 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Hall-Stoodley, L. et al. Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunol. Med. Microbiol. 65, 127–145 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Costerton, J. W., Geesey, G. G. & Cheng, K. J. How bacteria stick. Sci. Am. 238, 86–95 (1978).

    Article  CAS  PubMed  Google Scholar 

  20. Li, H. et al. Antibacterial activity of antibiotic coated silicone grafts. J. Urol. 160, 1910–1913 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Darouiche, R. O. Treatment of infections associated with surgical implants. N. Engl. J. Med. 350, 1422–1429 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Safdar, N. & Maki, D. G. Use of vancomycin-containing lock or flush solutions for prevention of bloodstream infection associated with central venous access devices: a meta-analysis of prospective, randomized trials. Clin. Infect. Dis. 43, 474–484 (2006).

    Article  PubMed  Google Scholar 

  23. Bjarnsholt, T., Tolker-Nielsen, T., Givskov, M., Janssen, M. & Chrsitensen, L. Detection of bacteria by FISH in culture-negative soft tissue filler lesions. Dermatol. Surg. 35, 1620–1624 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. 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 

  25. Bjarnsholt, T. et al. Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen. 16, 2–10 (2008).

    Article  PubMed  Google Scholar 

  26. Bjarnsholt, T. et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr. Pulmonol. 44, 547–558 (2009).

    Article  PubMed  Google Scholar 

  27. Fexby, S. et al. Biological Trojan horse: Antigen 43 provides specific bacterial uptake and survival in human neutrophils. Infect. Immun. 75, 30–34 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Fux, C. A., Costerton, J. W., Stewart, P. S. & Stoodley, P. Survival strategies of infectious biofilms. Trends Microbiol. 13, 34–40 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Stewart, P. S. New ways to stop biofilm infections. Lancet 361, 97 (2003).

    Article  PubMed  Google Scholar 

  30. Stewart, P. S. & Costerton, J. W. Antibiotic resistance of bacteria in biofilms. Lancet 358, 135–138 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Kim, J., Hahn, J. S., Franklin, M. J., Stewart, P. S. & Yoon, J. Tolerance of dormant and active cells in Pseudomonas aeruginosa PA01 biofilm to antimicrobial agents. J. Antimicrob. Chemother. 63, 129–135 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Walters, M. C. et al. 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 

  33. Hentzer, M., Eberl, L. & Givskov, M. Transcriptome analysis of Pseudomonas aeruginosa biofilm development: anaerobic respiration and iron limitation. Biofilms 2, 37–61 (2005).

    Article  Google Scholar 

  34. Marvig, R. L. et al. Mutations in 23S rRNA confer resistance against azithromycin in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 56, 4519–4521 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Imamura, Y. et al. Azithromycin exhibits bactericidal effects on Pseudomonas aeruginosa through interaction with the outer membrane. Antimicrob. Agents Chemother. 49, 1377–1380 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Saiman, L. The use of macrolide antibiotics in patients with cystic fibrosis. Curr. Opin. Pulm. Med. 10, 515–523 (2004).

    Article  PubMed  Google Scholar 

  37. Gillis, R. J. & Iglewski, B. H. Azithromycin retards Pseudomonas aeruginosa biofilm formation. J. Clin. Microbiol. 42, 5842–5845 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ciofu, O., Giwercman, B., Pedersen, S. S. & Høiby, N. Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center. APMIS 102, 674–680 (1994).

    Article  CAS  PubMed  Google Scholar 

  39. Roveta, S., Marchese, A. & Schito, G. C. Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp. Int. J. Antimicrob. Agents 31, 321–328 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Mascio, C. T., Alder, J. D. & Silverman, J. A. Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob. Agents Chemother. 51, 4255–4260 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. De Rosa, F. G. et al. Early experience with high-dosage daptomycin for prosthetic infections. Clin. Infect. Dis. 49, 1772–1773 (2009).

    Article  PubMed  Google Scholar 

  42. Hansen, C. R., Pressler, T. & Hoiby, N. Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonization in cystic fibrosis patients: 15 years experience. J. Cyst. Fibros. 7, 523–530 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Hurdle, J. G., O'Neill, A. J., Chopra, I. & Lee, R. E. Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nature Rev. Microbiol. 9, 62–75 (2011).

    Article  CAS  Google Scholar 

  44. Palmer, R. J. et al. in Biofilm Infections (eds Bjarnsholt, T., Moser, C., Jensen, P. O. & Høiby, N.) 35–60 (Springer, 2010).

    Google Scholar 

  45. Zimmerli, W. & Trampuz, A. in Biofilm Infections (eds Bjarnsholt, T., Moser, C., Jensen, P. O. & Høiby, N.) 91–110 (Springer, 2010).

    Google Scholar 

  46. Flemming, H. C., Neu, T. R. & Wozniak, D. J. The EPS matrix: the “house of biofilm cells”. J. Bacteriol. 189, 7945–7947 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Madsen, J. S., Burmolle, M., Hansen, L. H. & Sorensen, S. J. The interconnection between biofilm formation and horizontal gene transfer. FEMS Immunol. Med. Microbiol. 65, 183–195 (2012).

    Article  CAS  PubMed  Google Scholar 

  48. Christensen, B. B. et al. Molecular tools for study of biofilm physiology. Methods Enzymol. 310, 20–42 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Pamp, S. J., Sternberg, C. & Tolker-Nielsen, T. Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy. Cytometry A 75, 90–103 (2009).

    Article  PubMed  Google Scholar 

  50. Klausen, M. et al. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol. Microbiol. 48, 1511–1524 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Klausen, M., aes-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).

    Article  CAS  PubMed  Google Scholar 

  52. Schleheck, D. et al. Pseudomonas aeruginosa PAO1 preferentially grows as aggregates in liquid batch cultures and disperses upon starvation. PLoS ONE 4, e5513 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Oliver, J. D. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34, 415–425 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Folsom, J. P. et al. Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis. BMC Microbiol. 10, 294 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Alhede, M. et al. Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm. PLoS ONE 6, e27943 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Worlitzsch, D. et al. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Invest. 109, 317–325 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Aanaes, K. et al. Decreased mucosal oxygen tension in the maxillary sinuses in patients with cystic fibrosis. J. Cyst. Fibros. 10, 114–120 (2011).

    Article  PubMed  Google Scholar 

  59. Yang, L. et al. In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections. J. Bacteriol. 190, 2767–2776 (2008).

    Article  CAS  PubMed  Google Scholar 

  60. Lee, B. et al. Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J. Clin. Microbiol. 43, 5247–5255 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lee, B. et al. Mucoid Pseudomonas aeruginosa isolates maintain the biofilm formation capacity and the gene expression profiles during the chronic lung infection of CF patients. Acta. Pathol. Microbiol. Scand. Suppl. 119, 263–274 (2011).

    Article  Google Scholar 

  62. Ciofu, O., Mandsberg, L. F., Bjarnsholt, T., Wassermann, T. & Hoiby, N. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology 156, 1108–1119 (2010).

    Article  CAS  PubMed  Google Scholar 

  63. Folkesson, A. et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nature Rev. Microbiol. 10, 841–851 (2012).

    Article  CAS  Google Scholar 

  64. Moscoso, J. A., Mikkelsen, H., Heeb, S., Williams, P. & Filloux, A. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ. Microbiol. 13, 3128–3138 (2011).

    Article  CAS  PubMed  Google Scholar 

  65. Hickman, J. W., Tifrea, D. F. & Harwood, C. S. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc. Natl Acad. Sci. USA 102, 14422–14427 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Mikkelsen, H., Sivaneson, M. & Filloux, A. Key two-component regulatory systems that control biofilm formation in Pseudomonas aeruginosa. Environ. Microbiol. 13, 1666–1681 (2011).

    Article  CAS  PubMed  Google Scholar 

  67. Sivaneson, M., Mikkelsen, H., Ventre, I., Bordi, C. & Filloux, A. Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression. Mol. Microbiol. 79, 1353–1366 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Borlee, B. R. et al. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol. Microbiol. 75, 827–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Meissner, A. et al. Pseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL domain-dependent modulation of the intracellular level of cyclic diguanylate. Environ. Microbiol. 9, 2475–2485 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Petrova, O. E. & Sauer, K. SagS contributes to the motile-sessile switch and acts in concert with BfiSR to enable Pseudomonas aeruginosa biofilm formation. J Bacteriol. 193, 6614–6628 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Banin, E., Vasil, M. L. & Greenberg, E. P. Iron and Pseudomonas aeruginosa biofilm formation. Proc. Natl Acad. Sci. USA 102, 11076–11081 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Smith, D. J., Lamont, I. L., Anderson, G. J. & Reid, D. W. Targeting iron uptake to control Pseudomonas aeruginosa infections in cystic fibrosis. Eur. Respir. J. http://dx.doi.org/10.1183/09031936.00124012 (2012).

  73. Bagge, N. et al. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob. Agents Chemother. 48, 1175–1187 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Hoffman, L. R. et al. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436, 1171–1175 (2005).

    Article  CAS  PubMed  Google Scholar 

  75. Rybtke, M. T. et al. The implication of Pseudomonas aeruginosa biofilms in infections. Inflamm. Allergy Drug Targets. 10, 141–157 (2011).

    Article  CAS  PubMed  Google Scholar 

  76. Taga, M. E. & Bassler, B. L. Chemical communication among bacteria. Proc. Natl Acad. Sci. USA 100, 14549–14554 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Zhu, J. et al. Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc. Natl Acad. Sci. USA 99, 3129–3134 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Gilligan, P. H. Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4, 35–51 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Whiteley, M., Lee, K. M. & Greenberg, E. P. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 96, 13904–13909 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Eberl, L., Molin, S. & Givskov, M. Surface motility of serratia liquefaciens MG1. J. Bacteriol. 181, 1703–1712 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Eberl, L. et al. Involvement of N-acyl-l-homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens. Mol. Microbiol. 20, 127–136 (1996).

    Article  CAS  PubMed  Google Scholar 

  82. Kohler, T., Curty, L. K., Barja, F., van Delden, C. & Pechere, J. C. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J. Bacteriol. 182, 5990–5996 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Huber, B. et al. The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility. Microbiology 147, 2517–2528 (2001).

    Article  CAS  PubMed  Google Scholar 

  84. Magnuson, R., Solomon, J. & Grossman, A. D. Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell 77, 207–216 (1994).

    Article  CAS  PubMed  Google Scholar 

  85. Qin, X., Singh, K. V., Weinstock, G. M. & Murray, B. E. Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infect. Immun. 68, 2579–2586 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Hengzhuang, W., Wu, H., Ciofu, O., Song, Z. & Hoiby, N. Pharmacokinetics/pharmacodynamics of colistin and imipenem on mucoid and nonmucoid Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 55, 4469–4474 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Bink, A. et al. The non-steroidal anti-inflammatory drug diclofenac potentiates the in vivo activity of caspofungin against Candida albicans biofilms. J. Infect. Dis. 206, 1790–1797 (2012).

    Article  CAS  PubMed  Google Scholar 

  88. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999).

    Article  CAS  PubMed  Google Scholar 

  89. Costerton, J. W. et al. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41, 435–464 (1987).

    Article  CAS  PubMed  Google Scholar 

  90. Alipour, M., Suntres, Z. E. & Omri, A. Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa. J. Antimicrob. Chemother. 64, 317–325 (2009).

    Article  CAS  PubMed  Google Scholar 

  91. Stewart, P. S. Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob. Agents Chemother. 40, 2517–2522 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Herrmann, G. et al. Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa. J. Infect. Dis. 202, 1585–1592 (2010).

    Article  CAS  PubMed  Google Scholar 

  93. Borriello, G. et al. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob. Agents Chemother. 48, 2659–2664 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Yoon, S. S. et al. Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions. J. Clin. Invest. 116, 436–446 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. 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 

  96. Fernandez, L. & Hancock, R. E. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clin. Microbiol. Rev. 25, 661–681 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Dibdin, G. H., Assinder, S. J., Nichols, W. W. & Lambert, P. A. Mathematical model of β-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released β-lactamases. J. Antimicrob. Chemother. 38, 757–769 (1996).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Gillis, R. J. et al. Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 49, 3858–3867 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Chiang, W. C., Pamp, S. J., Nilsson, M., Givskov, M. & Tolker-Nielsen, T. The metabolically active subpopulation in Pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms. FEMS Immunol. Med. Microbiol. 65, 245–256 (2012).

    Article  CAS  PubMed  Google Scholar 

  101. Poole, K. Bacterial stress responses as determinants of antimicrobial resistance. J. Antimicrob. Chemother. 67, 2069–2089 (2012).

    Article  CAS  PubMed  Google Scholar 

  102. Kvist, M., Hancock, V. & Klemm, P. Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl. Environ. Microbiol. 74, 7376–7382 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. 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 

  104. Hengzhuang, W. et al. High β-lactamase levels change the pharmacodynamics of β-lactam antibiotics in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 57, 196–204 (2012).

    Article  CAS  PubMed  Google Scholar 

  105. Driffield, K., Miller, K., Bostock, J. M., O'Neill, A. J. & Chopra, I. Increased mutability of Pseudomonas aeruginosa in biofilms. J. Antimicrob. Chemother. 61, 1053–1056 (2008).

    Article  CAS  PubMed  Google Scholar 

  106. Ciofu, O., Riis, B., Pressler, T., Poulsen, H. E. & Høiby, N. Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation. Antimicrob. Agents Chemother. 49, 2276–2282 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Xu, K. D., Franklin, M. J., Park, C. H., McFeters, G. A. & Stewart, P. S. Gene expression and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms. FEMS Microbiol. Lett. 199, 67–71 (2001).

    Article  CAS  PubMed  Google Scholar 

  108. Bjedov, I. et al. Stress-induced mutagenesis in bacteria. Science 300, 1404–1409 (2003).

    Article  CAS  PubMed  Google Scholar 

  109. Lewis, K. Persister cells. Annu. Rev. Microbiol. 64, 357–372 (2010).

    Article  CAS  PubMed  Google Scholar 

  110. Macia, M. D., Perez, J. L., Molin, S. & Oliver, A. Dynamics of mutator and antibiotic-resistant populations in a pharmacokinetic/pharmacodynamic model of Pseudomonas aeruginosa biofilm treatment. Antimicrob. Agents Chemother. 55, 5230–5237 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Boles, B. R. & Singh, P. K. Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc. Natl Acad. Sci. USA 105, 12503–12508 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  112. Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A. & Collins, J. J. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130, 797–810 (2007).

    Article  CAS  PubMed  Google Scholar 

  113. Zhang, Q. et al. Acceleration of emergence of bacterial antibiotic resistance in connected microenvironments. Science 333, 1764–1767 (2011).

    Article  CAS  PubMed  Google Scholar 

  114. Hausner, M. & Wuertz, S. High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis. Appl. Environ. Microbiol. 65, 3710–3713 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Gristina, A. Biomaterial-centered infection: microbial adhesion versus tissue integration. 1987. Clin. Orthop. Relat. Res. 427, 4–12 (2004).

    Article  Google Scholar 

  116. Wahlig, H. & Buchholz, H. W. [Experimental and clinical studies on the release of gentamicin from bone cement]. Chirurg 43, 441–445 (1972).

    CAS  PubMed  Google Scholar 

  117. Stensballe, J. et al. Infection risk with nitrofurazone-impregnated urinary catheters in trauma patients: a randomized trial. Ann. Intern. Med. 147, 285–293 (2007).

    Article  PubMed  Google Scholar 

  118. O'Grady, N. P. et al. Guidelines for the prevention of intravascular catheter-related infections. Am. J. Infect. Control 39, S1–34 (2011).

    Article  PubMed  Google Scholar 

  119. Berra, L. et al. Internally coated endotracheal tubes with silver sulfadiazine in polyurethane to prevent bacterial colonization: a clinical trial. Intensive Care Med. 34, 1030–1037 (2008).

    Article  CAS  PubMed  Google Scholar 

  120. Kittinger, C. et al. Antimicrobial activity of gentamicin palmitate against high concentrations of Staphylococcus aureus. J. Mater. Sci. Mater. Med. 22, 1447–1453 (2011).

    Article  CAS  PubMed  Google Scholar 

  121. Crnich, C. J. & Maki, D. G. Are antimicrobial-impregnated catheters effective? When does repetition reach the point of exhaustion? Clin. Infect. Dis. 41, 681–685 (2005).

    Article  PubMed  Google Scholar 

  122. Cho, Y. H. et al. Prophylactic efficacy of a new gentamicin-releasing urethral catheter in short-term catheterized rabbits. BJU Int. 87, 104–109 (2001).

    Article  CAS  PubMed  Google Scholar 

  123. Gaonkar, T. A., Sampath, L. A. & Modak, S. M. Evaluation of the antimicrobial efficacy of urinary catheters impregnated with antiseptics in an in vitro urinary tract model. Infect. Control Hosp. Epidemiol. 24, 506–513 (2003).

    Article  PubMed  Google Scholar 

  124. Johnson, J. R., Kuskowski, M. A. & Wilt, T. J. Systematic review: antimicrobial urinary catheters to prevent catheter-associated urinary tract infection in hospitalized patients. Ann. Intern. Med. 144, 116–126 (2006).

    Article  PubMed  Google Scholar 

  125. Siddiq, D. M. & Darouiche, R. O. New strategies to prevent catheter-associated urinary tract infections. Nature Rev. Urol. 9, 305–314 (2012).

    Article  CAS  Google Scholar 

  126. Pickard, R. et al. Antimicrobial catheters for reduction of symptomatic urinary tract infection in adults requiring short-term catheterisation in hospital: a multicentre randomised controlled trial. Lancet 380, 1927–1935 (2012).

    Article  CAS  PubMed  Google Scholar 

  127. Vasilev, K., Cook, J. & Griesser, H. J. Antibacterial surfaces for biomedical devices. Expert. Rev. Med. Devices 6, 553–567 (2009).

    Article  PubMed  Google Scholar 

  128. Bak, J., Begovic, T., Bjarnsholt, T. & Nielsen, A. A UVC device for intra-luminal disinfection of catheters: in vitro tests on soft polymer tubes contaminated with Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans. Photochem. Photobiol. 87, 1123–1128 (2011).

    Article  CAS  PubMed  Google Scholar 

  129. Polo, A. et al. Effects of photoactivated titanium dioxide nanopowders and coating on planktonic and biofilm growth of Pseudomonas aeruginosa. Photochem. Photobiol. 87, 1387–1394 (2011).

    Article  CAS  PubMed  Google Scholar 

  130. Brady, R. A., O'May, G. A., Leid, J. G., Costerton, J. W. & Shirtliff, M. E. Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment. Infect. Immun. 79, 1797–1803 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Doring, G., Meisner, C. & Stern, M. A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc. Natl Acad. Sci. USA 104, 11020–11025 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Nilsson, E., Larsson, A., Olesen, H. V., Wejaker, P. E. & Kollberg, H. Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients. Pediatr. Pulmonol. 43, 892–899 (2008).

    Article  PubMed  Google Scholar 

  133. Frederiksen, B., Pressler, T., Hansen, A., Koch, C. & Hoiby, N. Effect of aerosolized rhDNase (Pulmozyme) on pulmonary colonization in patients with cystic fibrosis. Acta Paediatr. 95, 1070–1074 (2006).

    Article  PubMed  Google Scholar 

  134. Bjarnsholt, T. et al. Quorum sensing and virulence of Pseudomonas aeruginosa during lung infection of cystic fibrosis patients. PLoS ONE 5, e10115 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Alhede, M. et al. Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes. Microbiology 155, 3500–3508 (2009).

    Article  CAS  PubMed  Google Scholar 

  136. Jensen, P. O. et al. Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. Microbiology 153, 1329–1338 (2007).

    Article  CAS  PubMed  Google Scholar 

  137. van Gennip, M. et al. Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo. Infect. Immun. 80, 2601–2607 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Jackson, G. G., Dowling, H. F. & Lepper, M. H. Pathogenicity of staphylococci; a comparison of alpha-hemolysin production with the coagulase test and clinical observations of virulence. N. Engl. J. Med. 252, 1020–1025 (1955).

    Article  CAS  PubMed  Google Scholar 

  139. van Gennip, M. et al. Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes. APMIS 117, 537–546 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Bjarnsholt, T. et al. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology 151, 3873–3880 (2005).

    Article  CAS  PubMed  Google Scholar 

  141. Ciofu, O., Bagge, N. & Høiby, N. Antibodies against beta-lactamase can improve ceftazidime treatment of lung infection with beta-lactam-resistant Pseudomonas aeruginosa in a rat model of chronic lung infection. APMIS 110, 881–891 (2002).

    Article  CAS  PubMed  Google Scholar 

  142. Ciofu, O. Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response. APMIS Suppl. 116, 1–47 (2003).

    Google Scholar 

  143. Ciofu, O. et al. Antibodies against Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis are markers of the development of resistance of P. aeruginosa to beta-lactams. J. Antimicrob. Chemother. 35, 295–304 (1995).

    Article  CAS  PubMed  Google Scholar 

  144. Ciofu, O., Petersen, T. D., Jensen, P. & Hoiby, N. Avidity of anti-P aeruginosa antibodies during chronic infection in patients with cystic fibrosis. Thorax 54, 141–144 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Bjarnsholt, T. & Givskov, M. Quorum sensing inhibitory drugs as next generation antimicrobials: worth the effort? Curr. Infect. Dis. Rep. 10, 22–28 (2008).

    Article  PubMed  Google Scholar 

  146. Boles, B. R. & Horswill, A. R. agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS. Pathog. 4, e1000052 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Bjarnsholt, T. et al. In vitro screens for quorum sensing inhibitors and in vivo confirmation of their effect. Nature Protoc. 5, 282–293 (2010).

    Article  CAS  Google Scholar 

  148. Wu, H. et al. Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J. Antimicrob. Chemother. 53, 1054–1061 (2004).

    Article  CAS  PubMed  Google Scholar 

  149. Hentzer, M. et al. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87–102 (2002).

    Article  CAS  PubMed  Google Scholar 

  150. Skindersoe, M. E. et al. Quorum sensing antagonism from marine organisms. Mar. Biotechnol. 10, 56–63 (2008).

    Article  CAS  Google Scholar 

  151. Rasmussen, T. B. & Givskov, M. Quorum-sensing inhibitors as anti-pathogenic drugs. Int. J. Med. Microbiol. 296, 149–161 (2006).

    Article  CAS  PubMed  Google Scholar 

  152. Rasmussen, T. B. et al. Identity and effects of quorum sensing inhibitors produced by Penicillum species. Microbiology 151, 1325–1340 (2005).

    Article  CAS  PubMed  Google Scholar 

  153. Rasmussen, T. B. et al. Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. J. Bacteriol. 187, 1799–1814 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Camara, M., Williams, P. & Hardman, A. Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infect. Dis. 2, 667–676 (2002).

    Article  CAS  PubMed  Google Scholar 

  155. Hentzer, M. & Givskov, M. Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J. Clin. Invest. 112, 1300–1307 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Jakobsen, T. H. et al. Ajoene, a sulfur-rich molecule from garlic, inhibits genes controlled by quorum sensing. Antimicrob. Agents Chemother. 56, 2314–2325 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. 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 

  158. Christensen, L. D. et al. Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model. J. Antimicrob. Chemother. 67, 1198–1206 (2012).

    Article  CAS  PubMed  Google Scholar 

  159. Givskov, M. et al. Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. J. Bacteriol. 178, 6618–6622 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Ankri, S. & Mirelman, D. Antimicrobial properties of allicin from garlic. Microbes. Infect. 1, 125–129 (1999).

    Article  CAS  PubMed  Google Scholar 

  161. Song, Z. et al. Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection. Phytomedicine 17, 1040–1046 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Wu, H. et al. Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation. FEMS Immunol. Med. Microbiol. 62, 49–56 (2011).

    Article  CAS  PubMed  Google Scholar 

  163. Choi, K. T. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng C A Meyer. Acta Pharmacol. Sin. 29, 1109–1118 (2008).

    Article  CAS  PubMed  Google Scholar 

  164. Skindersoe, M. E. et al. Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 52, 3648–3663 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. Kohler, T., Guanella, R., Carlet, J. & van Delden, C. Quorum sensing-dependent virulence during Pseudomonas aeruginosa colonisation and pneumonia in mechanically ventilated patients. Thorax 65, 703–710 (2010).

    Article  PubMed  Google Scholar 

  166. Patriquin, G. M. et al. Influence of quorum sensing and iron on twitching motility and biofilm formation in Pseudomonas aeruginosa. J. Bacteriol. 190, 662–671 (2008).

    Article  CAS  PubMed  Google Scholar 

  167. 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 

  168. Budzikiewicz, H. Siderophore-antibiotic conjugates used as Trojan horses against Pseudomonas aeruginosa. Curr. Top. Med. Chem. 1, 73–82 (2001).

    Article  CAS  PubMed  Google Scholar 

  169. Ghosh, A. et al. Iron transport-mediated drug delivery using mixed-ligand siderophore-beta-lactam conjugates. Chem. Biol. 3, 1011–1019 (1996).

    Article  CAS  PubMed  Google Scholar 

  170. Reid, D. W. et al. Iron chelation directed against biofilms as an adjunct to conventional antibiotics. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L857–L858 (2009).

    Article  CAS  PubMed  Google Scholar 

  171. Moreau-Marquis, S., O'Toole, G. A. & Stanton, B. A. Tobramycin and FDA-approved iron chelators eliminate Pseudomonas aeruginosa biofilms on cystic fibrosis cells. Am. J. Respir. Cell. Mol. Biol. 41, 305–313 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Banin, E. et al. The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent. Proc. Natl Acad. Sci. USA 105, 16761–16766 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  173. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. Pichon, C. & Felden, B. Small RNA gene identification and mRNA target predictions in bacteria. Bioinformatics 24, 2807–2813 (2008).

    Article  CAS  PubMed  Google Scholar 

  175. Kint, G., De, C. D., Marchal, K., Vanderleyden, J. & De Keersmaecker, S. C. The small regulatory RNA molecule MicA is involved in Salmonella enterica serovar Typhimurium biofilm formation. BMC Microbiol. 10, 276 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. Bordi, C. & de Bentzmann, S. Hacking into bacterial biofilms: a new therapeutic challenge. Ann. Intensive Care 1, 19 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  177. Achermann, Y., Vogt, M., Leunig, M., Wust, J. & Trampuz, A. Improved diagnosis of periprosthetic joint infection by multiplex PCR of sonication fluid from removed implants. J. Clin. Microbiol. 48, 1208–1214 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  178. Rohacek, M. et al. Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture. Circulation 121, 1691–1697 (2010).

    Article  PubMed  Google Scholar 

  179. Sampedro, M. F. et al. A biofilm approach to detect bacteria on removed spinal implants. Spine 35, 1218–1224 (2010).

    Article  PubMed  Google Scholar 

  180. Bjerkan, G., Witso, E. & Bergh, K. Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthop. 80, 245–250 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  181. Kobayashi, N., Bauer, T. W., Tuohy, M. J., Fujishiro, T. & Procop, G. W. Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant. Clin. Orthop. Relat. Res. 457, 210–213 (2007).

    PubMed  Google Scholar 

  182. Young, D., Morton, R. & Bartley, J. Therapeutic ultrasound as treatment for chronic rhinosinusitis: preliminary observations. J. Laryngol. Otol. 124, 495–499 (2010).

    Article  CAS  PubMed  Google Scholar 

  183. Cui, H., Thomee, E., Noble, J. G., Reynard, J. M. & Turney, B. W. Efficacy of the lithotripsy in treating lower pole renal stones. Urolithiasis 41, 231–234 (2013).

    Article  PubMed  Google Scholar 

  184. Kaplan, J. B. Therapeutic potential of biofilm-dispersing enzymes. Int. J. Artif. Organs 32, 545–554 (2009).

    Article  CAS  PubMed  Google Scholar 

  185. Alkawash, M. A., Soothill, J. S. & Schiller, N. L. Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 114, 131–138 (2006).

    Article  CAS  PubMed  Google Scholar 

  186. Sauer, K. et al. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186, 7312–7326 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  187. Duoll, I. et al. An in vitro study of the rheology of cystic fibrosis sputum following treatment with oligoG alginate oligosaccharide. 23rd Annual North American Cystic Fibrosis Congress [online], (Anaheim, USA; 2011).

    Google Scholar 

  188. Davies, D. G. & Marques, C. N. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J. Bacteriol. 191, 1393–1403 (2009).

    Article  CAS  PubMed  Google Scholar 

  189. 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 

  190. Gjermansen, M., Ragas, P. & Tolker-Nielsen, T. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal. FEMS Microbiol. Lett. 265, 215–224 (2006).

    Article  CAS  PubMed  Google Scholar 

  191. Ma, Q., Yang, Z., Pu, M., Peti, W. & Wood, T. K. Engineering a novel c-di-GMP-binding protein for biofilm dispersal. Environ. Microbiol. 13, 631–642 (2011).

    Article  CAS  PubMed  Google Scholar 

  192. Antoniani, D., Bocci, P., Maciag, A., Raffaelli, N. & Landini, P. Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors. Appl. Microbiol. Biotechnol. 85, 1095–1104 (2010).

    Article  CAS  PubMed  Google Scholar 

  193. Heijerman, H., Westerman, E., Conway, S., Touw, D. & Doring, G. Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus. J. Cyst. Fibros. 8, 295–315 (2009).

    Article  CAS  PubMed  Google Scholar 

  194. Mermel, L. A. et al. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the Infectious Diseases Society of America. Clin. Infect. Dis. 49, 1–45 (2009).

    Article  CAS  PubMed  Google Scholar 

  195. Broom, J. et al. Ethanol lock therapy to treat tunnelled central venous catheter-associated blood stream infections: results from a prospective trial. Scand. J. Infect. Dis. 40, 399–406 (2008).

    Article  CAS  PubMed  Google Scholar 

  196. Barbaric, D., Curtin, J., Pearson, L. & Shaw, P. J. Role of hydrochloric acid in the treatment of central venous catheter infections in children with cancer. Cancer 101, 1866–1872 (2004).

    Article  CAS  PubMed  Google Scholar 

  197. Goss, C. H. et al. Phase 1 pharmacokinetic and safety study of intravenous ganiteTM (Gallium nitrate) in cystic fibrosis. (Abstract 230) Pediatr. Pulmonol. 46 (Suppl. 34), 294 (2011).

    Google Scholar 

  198. Hill, D. et al. Antibiotic susceptabilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. J. Clin. Microbiol. 43, 5085–5090 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  199. Kohler, T., Dumas, J. L. & van Delden, C. Ribosome protection prevents azithromycin-mediated quorum-sensing modulation and stationary-phase killing of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 51, 4243–4248 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  200. Allison, K. R., Brynildsen, M. P. & Collins, J. J. Heterogeneous bacterial persisters and engineering approaches to eliminate them. Curr. Opin. Microbiol. 14, 593–598 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  201. Shinde, R. B., Chauhan, N. M., Raut, J. S. & Karuppayil, S. M. Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A. Ann. Clin. Microbiol. Antimicrob. 11, 27 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  202. Alem, M. A. & Douglas, L. J. Prostaglandin production during growth of Candida albicans biofilms. J. Med. Microbiol. 54, 1001–1005 (2005).

    Article  CAS  PubMed  Google Scholar 

  203. Alem, M. A. & Douglas, L. J. Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans. Antimicrob. Agents Chemother. 48, 41–47 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  204. Ghalehnoo, Z. R., Rashki, A., Najimi, M. & Dominguez, A. The role of diclofenac sodium in the dimorphic transition in Candida albicans. Microb. Pathog. 48, 110–115 (2010).

    Article  CAS  PubMed  Google Scholar 

  205. Marquez, B. Bacterial efflux systems and efflux pumps inhibitors. Biochimie 87, 1137–1147 (2005).

    Article  CAS  PubMed  Google Scholar 

  206. Liu, Y., Yang, L. & Molin, S. Synergistic activities of an efflux pump inhibitor and iron chelators against Pseudomonas aeruginosa growth and biofilm formation. Antimicrob. Agents Chemother. 54, 3960–3963 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  207. Tirouvanziam, R. et al. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. Proc. Natl Acad. Sci. USA 103, 4628–4633 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  208. Hughes, K. A., Sutherland, I. W. & Jones, M. V. Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiology 144, 3039–3047 (1998).

    Article  CAS  PubMed  Google Scholar 

  209. Webb, J. S. et al. Cell death in Pseudomonas aeruginosa biofilm development. J. Bacteriol. 185, 4585–4592 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  210. Morello, E. et al. Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards treatment and prevention. PLoS ONE 6, e16963 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  211. Alemayehu, D. et al. Bacteriophages phiMR299-2 and phiNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. MBio. 3, e00029–e00012 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  212. Merabishvili, M. et al. Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. PLoS ONE 4, e4944 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  213. Moons, P., Werckx, W., Van Houdt, R., Aertsen, A. & Michiels, C. W. Resistance development of bacterial biofilms against bacteriophage attack. Commun. Agr. Appl. Biol. Sci. 71, 297–300 (2006).

    CAS  Google Scholar 

  214. Fu, W. et al. Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system. Antimicrob. Agents Chemother. 54, 397–404 (2010).

    Article  CAS  PubMed  Google Scholar 

  215. Domenech, M., Garcia, E. & Moscoso, M. In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases. Antimicrob. Agents Chemother. 55, 4144–4148 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  216. Meng, X. et al. Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis. Appl. Environ. Microbiol. 77, 8272–8279 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  217. Rolain, J. M., Fancello, L., Desnues, C. & Raoult, D. Bacteriophages as vehicles of the resistome in cystic fibrosis. J. Antimicrob. Chemother. 66, 2444–2447 (2011).

    Article  CAS  PubMed  Google Scholar 

  218. Ojeniyi, B., Birch-Andersen, A., Mansa, B., Rosdahl, V. T. & Hoiby, N. Morphology of Pseudomonas aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An electron microscopy study. APMIS 99, 925–930 (1991).

    Article  CAS  PubMed  Google Scholar 

  219. Willner, D. et al. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS ONE 4, e7370 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  220. Parracho, H. M., Burrowes, B. H., Enright, M. C., McConville, M. L. & Harper, D. R. The role of regulated clinical trials in the development of bacteriophage therapeutics. J. Mol. Genet. Med. 6, 279–286 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  221. Wright, A., Hawkins, C. H., Anggard, E. E. & Harper, D. R. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clin. Otolaryngol. 34, 349–357 (2009).

    Article  CAS  PubMed  Google Scholar 

  222. Brussow, H. Pseudomonas biofilms, cystic fibrosis, and phage: a silver lining? MBio 3, e00061–e00112 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  223. Hoffmann, N. et al. Azithromycin blocks quorum sensing and alginate polymer formation and increases the sensitivity to serum and stationary growth phase killing of P. aeruginosa and attenuates chronic P. aeruginosa lung infection in Cftr−/− mice. Antimicrob. Agents Chemother. 51, 3677–3687 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  224. Song, Z. et al. Effects of intratracheal administration of novispirin G10 on a rat model of mucoid Pseudomonas aeruginosa lung infection. Antimicrob. Agents Chemother. 49, 3868–3874 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  225. Brinch, K. S. et al. Plectasin shows intracellular activity against Staphylococcus aureus in human THP-1 monocytes and in a mouse peritonitis model. Antimicrob. Agents Chemother. 53, 4801–4808 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  226. Payne, D. J., Gwynn, M. N., Holmes, D. J. & Pompliano, D. L. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature Rev. Drug Discov. 6, 29–40 (2007).

    Article  CAS  Google Scholar 

  227. Hassett, D. J. et al. Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Adv. Drug Deliv. Rev. 54, 1425–1443 (2002).

    Article  CAS  PubMed  Google Scholar 

  228. Lu, C. D., Winteler, H., Abdelal, A. & Haas, D. The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa. J. Bacteriol. 181, 2459–2464 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  229. 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 

  230. 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).

    Article  CAS  PubMed  Google Scholar 

  231. Ceri, H. et al. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37, 1771–1776 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  232. Anderl, J. N., Franklin, M. J. & Stewart, P. S. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 44, 1818–1824 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  233. Goeres, D. M. et al. A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor. Nature Protoc. 4, 783–788 (2009).

    Article  CAS  Google Scholar 

  234. Zelver, N. et al. Measuring antimicrobial effects on biofilm bacteria: from laboratory to field. Methods Enzymol. 310, 608–628 (1999).

    Article  CAS  PubMed  Google Scholar 

  235. Werthen, M. et al. An in vitro model of bacterial infections in wounds and other soft tissues. APMIS 118, 156–164 (2010).

    Article  PubMed  Google Scholar 

  236. Merritt, J. H., Kadouri, D. E. & O'Toole, G. A. Growing and analyzing static biofilms. Curr. Protoc. Microbiol. http://dx.doi.org/10.1002/9780471729259.mc01b01s00 (2005).

  237. Kirketerp-Moller, K. et al. Distribution, organization, and ecology of bacteria in chronic wounds. J. Clin. Microbiol. 46, 2717–2722 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  238. Bjarnsholt, T., Tolker-Nielsen, T., Hoiby, N. & Givskov, M. Interference of Pseudomonas aeruginosa signalling and biofilm formation for infection control. Expert. Rev. Mol. Med. 12, e11 (2010).

    Article  CAS  PubMed  Google Scholar 

  239. Tanaka, G. et al. Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents: beta-lactams and fluoroquinolones. Chemotherapy 45, 28–36 (1999).

    Article  CAS  PubMed  Google Scholar 

  240. Vrany, J. D., Stewart, P. S. & Suci, P. A. Comparison of recalcitrance to ciprofloxacin and levofloxacin exhibited by Pseudomonas aeruginosa biofilms displaying rapid-transport characteristics. Antimicrob. Agents Chemother. 41, 1352–1358 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  241. Zhang, L. & Mah, T. F. Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. J. Bacteriol. 190, 4447–4452 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  242. Anwar, H., Strap, J. L. & Costerton, J. W. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob. Agents Chemother. 36, 1347–1351 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  243. Mulcahy, H., Charron-Mazenod, L. & Lewenza, S. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS. Pathog. 4, e1000213 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  244. Hocquet, D. et al. MexXY–OprM efflux pump is necessary for a adaptive resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother. 47, 1371–1375 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of International Health, Immunology and Microbiology, Costerton Biofilm Centre Faculty of Health Sciences, University of Copenhagen, Copenhagen N, 2200, Denmark

    Thomas Bjarnsholt, Oana Ciofu, Michael Givskov & Niels Høiby

  2. Department of Clinical Microbiology, Rigshospitalet, Copenhagen Ø, 2100, Denmark

    Thomas Bjarnsholt & Niels Høiby

  3. BioSys, Technical University of Denmark, Kongens Lyngby, 2800, Denmark

    Søren Molin

  4. Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 637551, Singapore

    Michael Givskov

Authors
  1. Thomas Bjarnsholt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oana Ciofu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Søren Molin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Givskov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Niels Høiby
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Bjarnsholt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

PowerPoint slides

Glossary

Persister phenomenon

The hypothesis that so-called persister bacteria exist in biofilms. These are a small subpopulation of dormant cells that are highly tolerant to killing by antibiotics and survive antibiotic treatment.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bjarnsholt, T., Ciofu, O., Molin, S. et al. Applying insights from biofilm biology to drug development — can a new approach be developed?. Nat Rev Drug Discov 12, 791–808 (2013). https://doi.org/10.1038/nrd4000

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd4000

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research