Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin

J Antimicrob Chemother. 2003 Oct;52(4):598-604. doi: 10.1093/jac/dkg397. Epub 2003 Sep 1.


Objectives: In Pseudomonas aeruginosa, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C12-HSL and C4-HSL. Previous studies suggested that C4-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C4-HSL. We wondered therefore whether the role of C4-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling.

Methods: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements.

Results: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals.

Conclusions: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 4-Butyrolactone / analogs & derivatives*
  • 4-Butyrolactone / antagonists & inhibitors*
  • 4-Butyrolactone / metabolism
  • Azithromycin / pharmacology*
  • Biofilms / drug effects
  • Biofilms / growth & development*
  • Cell Communication / drug effects*
  • Cell Communication / physiology
  • Humans
  • Pseudomonas aeruginosa*
  • Signal Transduction / drug effects


  • N-butyrylhomoserine lactone
  • Azithromycin
  • 4-Butyrolactone