Identification, characterization and determination of the mode of action of new broad applicable, small molecule inhibitors of Salmonella biofilm formation

Salmonella is one of the leading causes of food borne infections worldwide. 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 a wide range of environmental influences such as desiccation, antibiotics, disinfectants 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 of Salmonella. To identify new Salmonella biofilm inhibitors we conducted a high-throughput screening (using the ‘Calgary Biofilm device’) of a compound library, consisting 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. The library contains a broad range of compounds, selected on their possible drug ability. The compounds have a molecular weight between 200 and 500 Dalton and the screening was executed both at 16 °C and 37 °C, with a compound concentration between 20 µM and 50 µM. We aim at identifying compounds that inhibit biofilm formation, but that do not kill the bacteria. In this way, the development of resistance, which is a major drawback of classical antibiotics, is less likely. As such, these biofilm inhibitors could be a sustainable alternative in the production of safe and healthy food. Out of the 20.000 compounds tested, we identified 144 (0.72 %) possible biofilm inhibitors (‘hits’), of which 68 are active at 16 °C, 34 compounds at 37 °C, and 38 compounds are active at both temperatures. Subsequently, the dose-response relationship of the ‘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 destruction 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 determined, to select and optimize the most potent compounds. Of the most interesting compounds the ‘mode of action’ will be determined using, among others, reporter gene fusions. Therefore we created a library of 78 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 (general regulators, matrix production, motility...). One of the advantages of using reporter-fusions is the cheap, fast and ease-of-use so it 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. 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.Presentationstatus: publishe