New broad applicable small molecule inhibitors of Salmonella biofilmformation: identification, characterization and determination of their mode of action

A major difficulty in the prevention and treatment of Salmonella infections is the fact that Salmonella is able to form biofilms on various biotic and abiotic surfaces. Within these biofilms, Salmonella is protected against i.a.. antibiotics and the host immune system. As such, biofilm formation is an important survival strategy of Salmonella, both in- and outside the host. Therefore, the prevention and/or eradication of these biofilms could be an effective way to limit the spread, and prevent infections of Salmonella. To identify new Salmonella biofilm inhibitors we conducted a high-throughput screening of > 20.000 small molecules, in search of Salmonella biofilm inhibitors which are active at a temperature ranging from 16 °C to 37 °C, and therefore have potential to be used both in- and outside the host. Additionally we aim to identify compounds that specifically inhibit the biofilm formation, but do not kill the bacteria in order to prevent or slow down the development of resistance to these compounds. We identified 144 (0.72 %) possible biofilm inhibitors. Subsequently, the dose-response relationship of these ‘hits’ was determined, as well as the effect of the compounds on the planktonic growth of Salmonella, using a ‘bioscreen’ (Labsystems). The compounds with maximum biofilm inhibitory activity and minimal effect on planktonic growth were studied further, both with respect to prevention and eradication of biofilms from Salmonella Typhimurium and Pseudomonas aeruginosa, at different temperatures (16 °C, 25 °C, 30 °C and 37 °C). Using these results we identified 11 compound classes, from which analogues were purchased (± 20 analogues/family resulting in a library of 227 analogues). Using these analogues an early “structure-activity relationship” was delineated, to select and optimize the most potent compounds. Of the most interesting compounds the ‘mode of action’ will be determined using amongst others reporter gene fusions. Therefore we created a library of 81 GFP promoter fusions of important Salmonella biofilm genes, selected out of literature and in-house tests (Steenackers and Hermans et al., in press). This way we can quickly (semi-high throughput) identify the effect of the compounds on specific biofilm-related processes. One of the advantages of using reporter-fusions is that it is cheap, fast and easy to use and thus can be conducted for several compounds. This will yield valuable knowledge about the ‘mode of action’ of our compounds, and will also be used to select a compound class for more extensive studies using microarray analyses and mutant libraries. Reference: Steenackers, H.P.L.#, Hermans, K.#, Vanderleyden, J., De Keersmaecker, S.C.J. 2011. Salmonella Biofilms: an Overview on Occurrence, Structure, Regulation and Eradication. Food Res Int. In press, doi:10.1016/j.foodres.2011.01.038. # Equal contribution.Posterstatus: publishe