Screening And Activity Evaluation Of Gram-negative Bacteria Quorum Sensing Inhibitors

In bacteria, quorum sensing (QS) is a process of chemical communication involving theproduction, release, and subsequent detection of signaling molecules. QS regulates theproduction of key virulence factors in many pathogens. Compared with traditionalantibiotics, potential QS inhibitors (QSIs) inhibit the QS mechanism and attenuatevirulence without influencing bacterial growth. Targeting QS-regulated virulence is anovel approach to tackling antimicrobial resistance, which can be used to disarmpathogens in the host.The purpose of this study is to track the QSIs from the secondary metabolites in marinemicroorganisms and herbal extracts by effective screening systems and evaluate itsbiological activity. The QSIs from marine microorganisms extracts and herbal extracts arepotential agents for the treatment of bacterial infections.More than130kinds of marine fungal strains, actinomycetes and yeast strains wereisolated from the seawater and mud of Huangdao District in Qingdao. One fungal extractswas found to exhibit quorum sensing inhibitory activity in Chromobacterium violaceumCV026. The fragment of18S rDNA of QF046was compared with those of referencestrains deposited in GenBank (NCBI) and the results showed that the sequences of QF046exhibited high identity with Penicillium chrysogenum. Using a bioautographic TLC assay,preparative TLC, and HPLC analysis, the active compound LM05with quorum sensinginhibitory activity was isolated from the extracts of the active strain Penicillium sp.QF046. By means of spectroscopic method (1H NMR,13C NMR), the chemical structureof this compound was elucidated. It was the first time to report that this compound couldinterfere with C. violaceum quorum sensing system. This compound could reduceviolacein production in C. violaceum, without affecting bacterial growth in the effectiverange of concentration. Based on RT-PCR, the compound had a negative effect on theexpression of the key QS regulatory genes in C. violaceum.Herbal extracts derived from plants are known for their application in traditionalmedicine. Therefore, searching for new QSIs from herbal extracts, which are rich, diverse source of low toxic and bioactive compounds, is necessary. During the screening of morethan70kinds of herbal extracts, clove extract was found to inhibit QS-controlled geneexpression in Pseudomonas aeruginosa QSIS-lasI and Chromobacterium violaceumCV026biosensors. Using a bioautographic TLC assay, preparative TLC, and HPLCanalysis, eugenol, the major constituent of clove extract, exhibited QS inhibitory activity.Eugenol at sub-inhibitory concentrations inhibited the production of virulence factors,including elastase, biofilm formation and violacein. Pyocyanin production in PAO1wasreduced by up to56%with50μM eugenol. To exclude the influence of other QS systemsin P. aeruginosa, we used two Escherichia coli biosensors, E. coli MG4/pKDT17thatproduces LasR and contains the lasB promoter fused to lacZ and E. coli pEAL08-2thatproduces PqsR and contains the pqsA promoter fused to lacZ. Using these two biosensors,we confirmed that eugenol inhibited the las and pqs QS systems.Our data identified eugenol as a novel QS inhibitor and indicated the potential of plantphenolic compounds in the functional development of anti-QS activity. Comparingeugenol with its structural analogs of their anti-quorum sensing activity, we found thatthe anti-quorum sensing activity of phenolic hydroxy-substituted compounds and itseffect on elastase, pyocyanin, biofilm production was significantly weaker than eugenol.This study concluded that the phenolic hydroxyl group of eugenol is essential to itsanti-bacterial quorum sensing activity. This conclusion may have certain guidingsignificance for the study of anti-QS activity of plant phenolic compounds.The quorum sensing inhibitors have different mechanisms of action compared withtraditional antibiotics. They can inhibit bacterial QS regulated pathogenic behaviorwithout imposing harsh selective pressure on bacterial growth. So the probability ofbacterial resistance could be significantly reduced. Our study has an important theoreticalsignificance and application prospects for basic research. Meanwhile, it is believed thatthis work can provide more new ideas for combating with bacterial resistance andintroduce new methods for developing anti-QS active products.