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

<it>Objectives</it>: In <it>Pseudomonas aeruginosa</it>, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C<inf>12</inf>-HSL and C<inf>4</inf>-HSL. Previous studies suggested that C<inf>4</inf>-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 C<inf>4</inf>-HSL. We wondered therefore whether the role of C<inf>4</inf>-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. <it>Methods</it>: 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. <it>Results</it>: Wild-type strain PAO-BI, as a result of its reduced C<inf>4</inf>-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 <it>lasI</it> mutant (deficient in 3-oxo-C<inf>12</inf>-HSL) produced similar amounts of biofilm to the wild-type, a <it>rhlI</it> mutant (deficient in C<inf>4</inf>-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C<inf>4</inf>-HSL. Azithromycin, known to reduce the production of both 3-oxo-C<inf>12</inf>-HSL and C<inf>4</inf>-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals. <it>Conclusions</it>: Our results indicate that C<inf>4</inf>-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.