Inhibition of multidrug efflux as a strategy to prevent biofilm formation

Objectives:Wehave recentlyshownthat inactivation of anyof the multidrug efflux systemsof Salmonella results in loss of the ability to form acompetent biofilm. The aim of this studywasto determine themechanism linking multidrug efflux and biofilm formation, and to determine whether inhibition of efflux is a viable antibiofilm strategy. Methods: Mutants lackingcomponents of the AcrAB-TolC systemin Salmonella enterica serovar Typhimuriumwere investigated for their ability to aggregate, produce biofilm matrix components and form a biofilm. The potential for export ofabiofilm-relevant substrate via effluxpumpswasinvestigatedandexpression of genesthat regulate multidrug efflux and production of biofilm matrix components was measured. The ability of efflux inhibitors carbonyl cyanidem-chlorophenylhydrazone, chlorpromazine and phenyl-arginine-β-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 defectwas not related to changes in cellular hydrophobicity, aggregative ability or export of any biofilm-specific factor. The biofilm defect resulted fromtranscriptional repression of curli biosynthesis genes and consequent inhibition of production of curli. All three efflux inhibitors significantly reduced biofilm production in both static andflowbiofilm assays, although different concentrations of each inhibitorwere most active against each species. Conclusions: This workshowsthat both genetic inactivation andchemical inhibition of effluxpumpsresults in transcriptional repression of biofilm matrix components and a lack of biofilm formation. Therefore, inhibition of efflux is a promising antibiofilm strategy.