Inhibition of multidrug efflux as a strategy to prevent biofilm formation.

OBJECTIVES: We have recently shown that inactivation of any of the multidrug efflux systems of Salmonella results in loss of the ability to form a competent biofilm. The aim of this study was to determine the mechanism linking multidrug efflux and biofilm formation, and to determine whether inhibition of efflux is a viable antibiofilm strategy. METHODS: Mutants lacking components of the AcrAB-TolC system in Salmonella enterica serovar Typhimurium were investigated for their ability to aggregate, produce biofilm matrix components and form a biofilm. The potential for export of a biofilm-relevant substrate via efflux pumps was investigated and expression of genes that regulate multidrug efflux and production of biofilm matrix components was measured. The ability of efflux inhibitors carbonyl cyanide m-chlorophenylhydrazone, chlorpromazine and phenyl-arginine-ß-naphthylamide to prevent biofilm formation by Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus under static and flow conditions was assessed. RESULTS: Mutants of Salmonella Typhimurium that lack TolC or AcrB, but surprisingly not AcrA, were compromised in their ability to form biofilms. This defect was not related to changes in cellular hydrophobicity, aggregative ability or export of any biofilm-specific factor. The biofilm defect resulted from transcriptional repression of curli biosynthesis genes and consequent inhibition of production of curli. All three efflux inhibitors significantly reduced biofilm production in both static and flow biofilm assays, although different concentrations of each inhibitor were most active against each species. CONCLUSIONS: This work shows that both genetic inactivation and chemical inhibition of efflux pumps results in transcriptional repression of biofilm matrix components and a lack of biofilm formation. Therefore, inhibition of efflux is a promising antibiofilm strategy.

Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm.

OBJECTIVES: To investigate the contribution of multidrug efflux pump systems of Salmonella enterica serovar Typhimurium to the formation of a competent biofilm. METHODS: Biofilm formation by a wild-type strain and 10 efflux mutant strains was quantified using crystal violet biofilm assays and visualized using scanning electron microscopy. Curli expression was investigated qualitatively and quantitatively by measuring binding of the dye Congo red to polymerized curli and by comparative RT-PCR. RESULTS: All efflux mutants of Salmonella Typhimurium were compromised in their ability to form biofilms. Scanning electron microscopy images showed that the mutants were able to adhere to a surface but were unable to form a complex three-dimensional biofilm. Congo red assays demonstrated an inability of the efflux mutants to produce curli, a proteinaceous filament present on the cell surface and an essential component of the Salmonella biofilm extracellular matrix. Mutants expressed significantly less csgB or csgD than wild-type. Chemical inactivation of efflux in wild-type Salmonella Typhimurium with the efflux inhibitors (EIs) phenyl-arginine-ß-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and chlorpromazine also repressed biofilm formation. CONCLUSIONS: Our data demonstrates a link between all efflux systems of Salmonella Typhimurium and biofilm formation. Loss of functional efflux gives rise to a lack of curli expression. Biofilm formation was also inhibited by addition of a variety of EIs with differing mechanisms of action, suggesting a novel role for EIs as anti-biofilm compounds.