Suppression of bacterial cell-cell signalling, biofilm formation and type III secretion system by citrus flavonoids.

AIM: This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. METHODS AND RESULTS: Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell-cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell-cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. CONCLUSION: The results of the study indicate a potential modulation of bacterial cell-cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell-cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell-cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.

Influence of autoinducer-2 (AI-2) and beef sample extracts on E. coli O157:H7 survival and gene expression of virulence genes yadK and hhA.

Bacterial cell-to-cell communication is mediated by autoinducer (AI) molecules such as AI-2 and has been reported to regulate gene expression in Escherichia coli O157:H7. We have previously shown that ground beef contains compounds that can inhibit sensing of AI-2 like activity. The hypothesis of this study was that AI-2 activity observed in conditioned medium (CM) will enhance E. coli O157:H7 survival and expression of virulence genes, whereas compounds inhibitory (such as those present in ground beef extracts) to AI-2 activity will negate these effects. E. coli O157:H7 luxS mutant strain VS 94 (incapable of synthesizing AI-2) was employed in these studies. The survival of this enteric bacterial pathogen as a function of AI-2 activity and the presence of AI-2 inhibitory compounds was studied at 4 degrees C. The number of survivors in the presence of AI-2 was significantly higher compared to the absence of AI-2, and the addition of ground beef extracts to conditioned medium negated the influence of AI-2 activity. Autoinducer AI-2 upregulated selected genes virulence genes (yadK, and hha), whereas the ground beef extract reversed the effect of AI-2 on the expression of the selected genes.

Identification of poultry meat-derived fatty acids functioning as quorum sensing signal inhibitors to autoinducer-2 (AI-2).

Autoinducer-2 (AI-2) is a compound that plays a key role in bacterial cell-to-cell communication (quorum sensing). Previous research has shown certain food matrices inhibit this signaling compound. Using the reporter strain, Vibrio harveyi BB170, quorum-sensing inhibitors contained in poultry meat wash (PMW) samples were characterized by molecular weight and hydrophobic properties using liquid chromatography systems. Most fractions that demonstrated AI-2 inhibition were 13.7 kDa or less, and had hydrophobic properties. Hexane was used to extract inhibitory compounds from a PMW preparation and the extract was further separated by gas chromatography (GC). Several fatty acids were identified and quantified. Linoleic acid, oleic acid, palmitic acid, and stearic acid were each tested for inhibition at 0.1, 1, and 10 mM concentrations. All samples expressed AI-2 inhibition (ranging from approximately 25% to 99%). Fatty acids, combined in concentrations equivalent to those determined by GC analysis, expressed inhibition at 59.5%, but higher combined concentrations (10- and 100-fold) had inhibition at 84.4% and 69.5%, respectively. The combined fatty acids (100-fold) did not demonstrate a substantial decrease in colony plate counts, despite presenting high AI-2 inhibition. These fatty acids, through modulating quorum sensing by inhibition, may offer a unique means to control foodborne pathogens and reduce microbial spoilage.