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

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. UV-radiation, 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, and prevent infections of Salmonella. To identify new Salmonella biofilm inhibitors a high-throughput screening of > 20,000 small molecules was conducted, 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. In total 144 (0.72 %) possible biofilm inhibitors were identified. 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. This way 3 chemically diverse compound families were selected (Robijns et al., 2012). Of these compound families the 'mode of action' is currently being 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 knowledge (Steenackers et al., 2012). By following the difference in fluorescence, i.e. gene-expression, with and without compound present, we can quickly (semi-high throughput) identify the effect of the compounds on specific biofilm-related processes. On