ASM Conference on Biofilms edition:5 location:Cancun, Mexico date:15-19 November 2009
A major difficulty in the control of Salmonella infections is the fact that Salmonella can form biofilms, in which it is protected against the influence of antibiotics, disinfectants and the immune system. Therefore, the prevention of biofilms could be an effective way to restrict the spread of Salmonella. We chemically synthesized different libraries of natural product analogues and tested their influence on Salmonella biofilm formation by using a high throughput biofilm assay. In this way we identified different classes of potent Salmonella biofilm inhibitors for which we delineated structure activity relationships. One of these classes are the brominated furanones, which were originally isolated from the seaweed Delisea pulchra and which are known to inhibit biofilm formation and quorum sensing (QS) in several other pathogens. The most interesting furanones are (Z)-4-bromo-5-(bromomethylene)-3-alkyl-2(5H)-furanones with chain lengths of two to six carbon atoms. We are currently optimizing the structure of the identified inhibitors by using computational QSAR models. Ligand based pharmacophore models are being used to identify new classes of inhibitors via scaffold hopping. We investigated the mode of action of the identified classes of biofilm inhibitors by using different complementary approaches, as exemplified by the case of the brominated furanones. To determine whether the brominated furanones inhibit biofilm formation by interfering with the quorum sensing systems of Salmonella, we studied the expression of the known target genes srgE and lsrA of SdiA and AI-2, respectively, in the presence and absence of furanones, by using gene reporter fusions. Surprisingly, no evidence was found that furanones act on the SdiA or AI-2 QS system of Salmonella. A microarray study was performed to analyze the gene expression profiles of Salmonella in the presence of (Z)-4-bromo-5-(bromomethylene)-3-ethyl-2(5H)-furanone. 130 genes were differentially expressed. The induced genes include genes that are involved in metabolism, stress response and drug sensitivity. Most of the repressed genes are involved in metabolism, the type III secretion system and flagellar biosynthesis. Flagella staining and motility assays confirmed that the furanone interferes with the synthesis of flagella. Since it has been shown that functional flagella are necessary for Salmonella biofilm formation (a flhD mutant does not form a biofilm in our test conditions), it is possible that interference with the flagellar assembly causes the observed biofilm defect. To enhance the efficacy of the biofilm inhibitors, we studied synergy with antibiotics. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. Conclusively, we identified several classes of potent Salmonella biofilm inhibitors and made progress in the elucidation of their mode of action.