Identification of new Salmonella biofilm inhibitors through a 'bottom-up' and 'top-down' approach.

A major difficulty in the control of Salmonella</>, and by extension most bacterial species, is the fact that they are able to develop multicellular, surface associated structures, termed biofilms. In these biofilms the bacteria are engulfed in a self-produced, gel-like matrix and are protected against many different stress factors, including antibiotics, disinfectants and immune defenses. Therefore, the main objective of this thesis was the identification of new and effective anti-biofilm compounds. Since bacteria can develop biofilms in virtually all environments and the regulation and composition of the biofilm(matrix) differs depending on these environments, new inhibitors should be broadly applicable, in different environments, at different temperatures and against several bacterial species. And last but not least, all inhibitors should only exert a biofilm specific activity, without affecting the (planktonic) growth of the bacteria. In this way the selective pressure reduces, thereby slowing down or preventing resistance development against these inhibitors.
 
For the identification of the broadly applicable and biofilm specific compounds, two compound libraries were screened. The most extensive library, which provides the bulk of this thesis, was delivered by the Centre for Drug Design and Discovery (CD3) and contained > 20,000 small-molecules. The library was screened for the prevention of Salmonella</> biofilms, both at 16 °C and 37 °C, and resulted in the initial identification of 140 hits. After further confirmation and characterization, 11 families with promising and broad applicable biofilm specific effects were selected for a first structure-activity relationship. The three most promising compound families were subsequently studied in more detail, against a total of nine different bacterial species.
A second, less extensive, library screened in this work contained a total of 672 synthetic peptides, synthesized based on naturally isolated peptides, mostly originating from tissue- and body-fluids of human and mice. The screening resulted in a total of fivepeptides showing promising properties for the further development into (specific) anti-biofilm strategies.
 
In addition to the identification and characterization of the activity, the mode of action (MOA) of the new biofilm inhibitors was determined. This provides valuable knowledge that can be used for further optimization of the activity and specificity of the compounds. Determination of the mode of action can also yield important insights about fundamental biofilm processes, which can be used for the development of additional anti-biofilm strategies.
Since the regulation and composition of bacterial biofilms varies depending on the growth environment, it is essential that these MOA studies are conducted in several conditions and in a time-lapse manner. Therefore,a reporter fusion library, a fast and easy accessible assay for MOA studies against Salmonella biofilms, was developed in this thesis. This GFPbased library currently contains 78 promoter fusions of important biofilm related genes, including genes involved in matrix production, motility, virulence, quorum sensing and metabolic processes.
This reporter fusion library was used to determine the mode of action, in four conditions (at 16 °C and 37 °C, both in standing and shaking conditions), of the two most promising small-molecule families. This revealed i.a.inhibitory effects on the Salmonella</> (and E. coli</>) biofilm master regulator CsgD, either via prevention of csgD</> transcription, or interference with the translation or function of theprotein. In both cases this resulted in the down-regulation of csgB</> (involved in curli fimbriae biosynthesis) and adrA</> expression (regulator of cellulose biosynthesis). Both curli fimbriae and cellulose are important components of the Salmonella biofilm matrix and their inhibition is likely to be one of the major factors involved in the MOA of the small-molecules. Additionally, several attachment related processes and motility and virulence regulators were affected by the compounds. 
Both compound families also induced important changes inthe expression of metabolic genes, such as reduction in iron-uptake mechanisms or changes in nitrogen metabolism. This is either a result of the direct effect of the compound (by blocking certain metabolic pathways), or a result of the reduced biofilm formation which therefore changes the metabolic need of the bacteria. Further study is needed to determine the exact cause and effect.
Nonetheless, all compounds seem to affectat least two pathways in the Salmonella</> biofilm formation, therefore additionally reducing the chance of resistance development.
 Conclusively, in this thesis different classes of potent bacterial biofilm inhibitors were identified, which show promise for further optimization in several different application fields. Mode of action studies revealed several pathways involved in the biofilm inhibition,including repression of the CsgD biofilm master regulator and attachment processes, coupled with changes in motility and virulence related process and the bacterial metabolism. 

Keywords:Salmonella, Biofilm, Biofilm inhibitors, Molecule library

Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Microbiology, Systems biology, Laboratory medicine

Project type:PhD project