Bee products (propolis, bee pollen and honey) as quorum sensing inhibitors in Chromobacterium violaceum (#229)
In opportunistic bacterial pathogens, especially in Gram-negative bacteria, inhibition of the quorum sensing system is an interesting strategy to overcome infection and resistance against antibiotics through suppressing bacterial virulence. The objective of this study was to elucidate the potential compound(s) in bee products (bee pollen, honey and propolis) that exhibits significant quorum sensing inhibitory (QSI) properties on Chromobacterium violaceum ATCC 12472 at the phenotypic and molecular levels. Preliminary screening on C. violaceum lawn agar revealed that manuka propolis had the strongest QSI activity, bee pollen had no detectable QSI activity and honey had bactericidal activity, likely due to a high sugar content. Thus, manuka propolis was fractionated using column chromatography and pooled via thin-layer chromatography (TLC). The pooled fractions were rescreened for the highest QSI effects in dose-dependent and time-dependent manners on C. violaceum lawn agars. One pooled fraction, PF5, showed the largest violacein inhibition zone (24.5 ± 2.5 mm) at 1 mg per disc. For vioA (a QS-regulated gene involved in violacein biosynthesis) expression assays, RNA were isolated from bacterial cultures after 24 hours of incubation with 300-500 µg/ml of PF5 and subjected to reverse-transcription real-time PCR. Interestingly, vioA was significantly down-regulated with incubation with at least 450 µg/ml of PF5. Phytochemical content analysis of manuka propolis, together with high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) of PF5 revealed a major composition of hydroxycinnamic acid-derivatives, where a compound with [M-H] of 247 was identified as isoprenyl caffeate. This compound is hypothesized as a competitive inhibitor of acyl homoserine lactone, therefore prevent the activation of QS mechanism in C. violaceum, and also possibly other Gram-negative bacteria. This is the first study to elucidate an active QSI compound in manuka propolis (isoprenyl caffeate), as well as to correlate phenotypical QSI observations in C. violaceum withthe molecular involvement of the main QS pathway of this bacterium. Further investigations on PF5 are recommended towards the development of potential propolis-based anti-pathogenic drugs.