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Figure 3: Examples of results from a QS screening. In a first step (A), the effect of a series of extracts from single compounds and mixes were tested for their capacity to inhibit a specific QS signal. In a second experiment (B), (mixes of) selected botanical extracts were further tested in a dilution experiment. The MIC value (red X signs) and the concentration at which the QS is reduced to 50% (blue hexagons) are given for three test products.


A


B


Apart from the role of QS signalling in human diseases, knowledge


about its relevance for veterinary pathogens, such as Clostridium perfringens, Yersinia pseudotuberculosis and Salmonella enterica, is also expanding. In addition, modulating QS in the gastro-intestinal tract has sparked interest as an approach to control gut microbial activity and composition, thereby influencing animal health and zootechnical performance.


QS as a tool to select ingredients We have used QS as a tool to select for highly active bioactive compounds when developing a new feed additive for broilers. Starting from botanicals and other raw materials for which we already had evidence that they have a positive effect on digestion, anti-oxidation and immunomodulation, we were left with still a long list of potential ingredients to choose from. As we wanted these new additives to have a significant activity on gut microbial activity and composition as well, we relied on QS assays to define the final composition of this novel phytogenic product. In a first step (Figure 3), we screened botanical ingredients,


individually and in mixes, for their capacity to inhibit two types of QS. This was achieved by making extracts of these ingredients, determining their MIC values for the different reporter strains, and adding them to a culture of these strains at concentrations well-below their MICs. Subsequently, the QS-dependent readout of the reporter strain (e.g. a fluorescent signal that is produced when QS is active) was compared with the readout of the strain after they had been incubated with the extracts. In a next step, we selected the most performant extracts and


made a dilution series for further testing of QS inhibition at even lower concentrations (Figure 3). Of note, we observed that the capacity to inhibit QS was not correlated with the MIC values of these substances.


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As can be seen in Figure 3B, “botanical 34” had the highest MIC compared to the other two test compounds, but the lowest concentration at which the QS signal was reduced by half. Based on these results, we selected a botanical mixture to test it


in a simple in vivo model: microscopic roundworms (C. elegans) that were infected with Salmonella typhimurium. It is important to note that we didn’t do this to evaluate the potential of the botanical prototype to reduce Salmonella colonization in broilers. Rather, we exploited the knowledge that Salmonella is able to colonize the digestive tract of C. elegans, culminating in QS-dependent production of toxins that affect viability of these roundworms.


Figure 4: C. elegans roundworms were grown in absence (grey) or in presence (orange) of Salmonella bacteria. After intestinal colonization, Salmonella will activate QS signals, culminating in the production of toxins, thereby decreasing C. elegans survival. When a botanical extract was added to the infected roundworms at a concentration not affecting Salmonella growth, it significantly reduced the Salmonella-induced mortality.


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