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

To investigate the contribution of multidrug efflux pump systems of Salmonella enterica serovar Typhimurium to the formation of a competent biofilm. 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 RTPCR. 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--naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and chlorpromazine also repressed biofilm formation. 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.