Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro
The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential QS inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems have been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of two of the major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.
Key wordsQuorum sensing inhibitor Halogenated furanones QSI monitor screen DNA microarray In vitro continuous-culture biofilm flow cell system Confocal scanning laser microscopy
We acknowledge the contributions made by Drs. Morten Hentzer, Hong Wu, Thomas Bovbjerg Rasmussen, Jens Bo Andersen, and Allan Bech Christensen with regard to the initial developments of the model systems. We are greateful for the support we have received over the years from the Lundbeck foundation, the Danish Strategical research Council, the German CF organization, Kirsten & Freddy foundation, the Villum foundation and Novoseeds to MG.
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