Quorum Sensing pp 253-263 | Cite as

Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro

  • Tim Holm Jakobsen
  • Maria van Gennip
  • Louise Dahl Christensen
  • Thomas Bjarnsholt
  • Michael Givskov
Part of the Methods in Molecular Biology book series (MIMB, volume 692)


The formation of biofilms in conjunction with quorum sensing (QS)-regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential quorum sensing inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of the two major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray-based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.

Key words

Quorum sensing inhibitor Halogenated furanones QSI monitor screen DNA microarray In vitro continuous-culture biofilm flow cell system Confocal Scanning Laser Microscopy 



We acknowledge the contributions made by Drs. Morten Hentzer, Hong Wu, Thomas Bovbjerg Rasmussen, Jens Bo Andersen, and Allan Bech Christensen with regard to the initial developments of the model systems.


  1. 1.
    Lewenza, S., Conway, B., Greenberg, E. P. and Sokol, P. A. (1999) Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI, J Bacteriol. 181, 748–756.PubMedGoogle Scholar
  2. 2.
    Bjarnsholt, T., Jensen, P. O., Burmolle, M., Hentzer, M., Haagensen, J. A., Hougen, H. P., et al. (2005) Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent, Microbiology.151, 373–383.PubMedCrossRefGoogle Scholar
  3. 3.
    Costerton, J. W., Stewart, P. S. and Greenberg, E. P. (1999) Bacterial biofilms: a common cause of persistent infections, Science. 284, 1318–1322.PubMedCrossRefGoogle Scholar
  4. 4.
    de Kievit, T. R. and Iglewski, B. H. (2000) Bacterial quorum sensing in pathogenic relationships, Infect Immun. 68, 4839–4849.PubMedCrossRefGoogle Scholar
  5. 5.
    Hentzer, M., Riedel, K., Rasmussen, T. B., Heydorn, A., Andersen, J. B., Parsek, M. R., et al. (2002) Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound, Microbiology. 148, 87–102.PubMedGoogle Scholar
  6. 6.
    Hentzer, M., Wu, H., Andersen, J. B., Riedel, K., Rasmussen, T. B., Bagge, N., et al. (2003) Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors, EMBO J. 22, 3803–3815.PubMedCrossRefGoogle Scholar
  7. 7.
    Wu, H., Song, Z., Hentzer, M., Andersen, J. B., Molin, S., Givskov, M., et al. (2004) Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice, J Antimicrob Chemother. 53, 1054–1061.PubMedCrossRefGoogle Scholar
  8. 8.
    Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., et al. (1996) Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling, J Bacteriol. 178, 6618–6622.PubMedGoogle Scholar
  9. 9.
    Rasmussen, T. B., Skindersoe, M. E., Bjarnsholt, T., Phipps, R. K., Christensen, K. B., Jensen, P. O., et al. (2005) Identity and effects of quorum-sensing inhibitors produced by Penicillium species, Microbiology. 151, 1325–1340.PubMedCrossRefGoogle Scholar
  10. 10.
    Bjarnsholt, T., Jensen, P. O., Rasmussen, T. B., Christophersen, L., Calum, H., Hentzer, M., et al. (2005) Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections, Microbiology. 151, 3873–3880.PubMedCrossRefGoogle Scholar
  11. 11.
    Rasmussen, T. B., Bjarnsholt, T., Skindersoe, M. E., Hentzer, M., Kristoffersen, P., Kote, M., et al. (2005) Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector, J Bacteriol. 187, 1799–1814.PubMedCrossRefGoogle Scholar
  12. 12.
    Skindersoe, M. E., Ettinger-Epstein, P., Rasmussen, T. B., Bjarnsholt, T., de Nys, R. and Givskov, M. (2008) Quorum sensing antagonism from marine organisms, Mar Biotechnol (NY). 10, 56–63.CrossRefGoogle Scholar
  13. 13.
    Muh, U., Schuster, M., Heim, R., Singh, A., Olson, E. R. and Greenberg, E. P. (2006) Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen, Antimicrob Agents Chemother. 50, 3674–3679.PubMedCrossRefGoogle Scholar
  14. 14.
    Amara, N., Mashiach, R., Amar, D., Krief, P., Spieser, S. A., Bottomley, M. J., et al. (2009) Covalent inhibition of bacterial quorum sensing, J Am Chem Soc. 131, 10610–10619.PubMedCrossRefGoogle Scholar
  15. 15.
    Riedel, K., Kothe, M., Kramer, B., Saeb, W., Gotschlich, A., Ammendola, A., et al. (2006) Computer-aided design of agents that inhibit the cep quorum-sensing system of Burkholderia cenocepacia, Antimicrob Agents Chemother. 50, 318–323.PubMedCrossRefGoogle Scholar
  16. 16.
    Persson, T., Hansen, T. H., Rasmussen, T. B., Skinderso, M. E., Givskov, M. and Nielsen, J. (2005) Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic, Org Biomol Chem. 3, 253–262.PubMedCrossRefGoogle Scholar
  17. 17.
    Persson, T., Givskov, M. and Nielsen, J. (2005) Quorum sensing inhibition: targeting chemical communication in gram-negative bacteria, Curr Med Chem. 12, 3103–3115.PubMedCrossRefGoogle Scholar
  18. 18.
    Hjelmgaard, T., Persson, T., Rasmussen, T. B., Givskov, M. and Nielsen, J. (2003) Synthesis of furanone-based natural product analogues with quorum sensing antagonist activity, Bioorg Med Chem. 11, 3261–3271.PubMedCrossRefGoogle Scholar
  19. 19.
    Olsen, J. A., Severinsen, R., Rasmussen, T. B., Hentzer, M., Givskov, M. and Nielsen, J. (2002) Synthesis of new 3- and 4-substituted analogues of acyl homoserine lactone quorum sensing autoinducers, Bioorg Med Chem Lett. 12, 325–328.PubMedCrossRefGoogle Scholar
  20. 20.
    Yang, L., Rybtke, M. T., Jakobsen, T. H., Hentzer, M., Bjarnsholt, T., Givskov, M., et al. (2009) Computer-aided identification of recognized drugs as Pseudomonas aeruginosa quorum-sensing inhibitors, Antimicrob Agents Chemother. 53, 2432–2443.PubMedCrossRefGoogle Scholar
  21. 21.
    Bjarnsholt, T., Jensen, P. O., Burmølle, M., Hentzer, M., Haagensen, J. A., Hougen, H. P., et al. (2005) Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent, Microbiology. 151, 373–383.PubMedCrossRefGoogle Scholar
  22. 22.
    Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M. and Molin, S. (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria, Appl Environ Microbiol. 64, 2240–2246.PubMedGoogle Scholar
  23. 23.
    Christensen, B. B., Sternberg, C., Andersen, J. B., Palmer, R. J., Jr., Nielsen, A. T., Givskov, M., et al. (1999) Molecular tools for study of biofilm physiology, Methods Enzymol. 310, 20–42.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Tim Holm Jakobsen
    • 1
  • Maria van Gennip
    • 1
  • Louise Dahl Christensen
    • 1
  • Thomas Bjarnsholt
    • 1
    • 2
  • Michael Givskov
    • 1
  1. 1.Department of International Health, Immunology, and Microbiology, Faculty of Health SciencesUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Clinical MicrobiologyRigshospitaletCopenhagenDenmark

Personalised recommendations