Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro

Abstract

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.

Paecilomycone Inhibits Quorum Sensing in Gram-Negative Bacteria

“…The experiments were performed as previously described ( 63 ). In brief, P. aeruginosa PAO1 reporter lines were grown overnight in AB minimal medium supplemented with 0.5% glucose and 0.5% Casamino Acids.…”

Gregatins, a Group of Related Fungal Secondary Metabolites, Inhibit Aspects of Quorum Sensing in Gram-Negative Bacteria

“…The experiments were performed as previously described (Jakobsen et al, 2018 ). In brief, overnight grown cultures were diluted in PBS to an OD 450 of 0.1–0.2 before addition to a 96-well plate with dilution ranges of compounds up to a volume of 200 μl (1:1, v:v).…”

“…C. violaceum was inoculated on tryptic soy agar (TSA), and single colonies were grown in tryptic soy broth (TSB) at 27 • C. PAO1 strains were inoculated on Luria agar (LA) at 37 • C, and single colonies were grown in AB minimal medium supplemented with 0.5% glucose and 0.5% casamino acids (Jakobsen et al, 2018), unless stated otherwise.…”

Ultrasound‐mediated therapies for the treatment of biofilms in chronic wounds: a review of present knowledge

“…By incapacitating this signalling mechanism, the regulation of gene expression, essential metabolic processes and virulence can be irreparably altered (Khmel, 2006). Natural and synthetic inhibitors of quorum sensing, such as furanones and Manuka honey, work by downregulating four major quorum sensing genes, which in turn has downstream consequences for genes associated with the biofilm phenotype (Wang et al, 2012;Jakobsen et al, 2018). However, it is important to note this is principally a method to render a biofilm more inert; it does not offer an immediate solution to detachment or physical eradication (LaSarre and Federle, 2013).…”