Study On Anti-quorum Sensing Active And Secondary Metabolites Of Marine Sediment Actinomycetes

In recent years,due to the excessive dependence and abuse of antibiotics in the medical field,most pathogens gradually develop drug resistance to traditional antibiotics.Therefore,solving bacterial resistance has become a major problem in the medical field.Many researches showed that the quorum sensing system regulated the toxin synthesis and biofilm formation of pathogens,and the quorum sensing inhibitors could regulate the pathogenic effect of pathogens by mediating the quorum sensing system of pathogens,so that pathogens would not produce resistance mutation.Therefore,the search for natural quorum sensing inhibitors is expected to prevent or coordinate the prevention and treatment of drug-resistant bacteria.The main purpose of this study was to isolate compounds with anti quorum sensing activity from the natural products of marine actinomycetes.121 strains of actinomycetes were isolated from the sediment samples of Dalian coastal waters by plate dilution method.Chromobacterium violaceum 12472 was used as the report strain to measure the anti-quorum sensing activity of isolated actinomycetes strains by using dics diffusion assay.Several multidrug resistant strains,including Staphylococcus aureus ATCC 25923、ATCC 43300,Enterococcus faecalis ATCC 51299 and Escherichia coli were used as the target bacteria to further test of the antibacterial activity of active isolates.Three strains(DL-99,DL-105,DL-106)with better activity were screened out and sequenced for 16S rDNA.Combined with their morphological characteristics,three active strains were identified as Streptomyces chyphonensis DL-99,Streptomyces griseoinarnatus DL-105 and Nocardiopsis dassonvillei DL-106,respectively.These three strains were cultured in different media to compare their effects on metabolites.Combined with the results of anti-quorum sensing activity,thin layer chromatography(TLC)analysis and high performance liquid chromatography(HPLC)analysis,YMS medium was regarded as the best culture medium for strain DL-106 and was selected for large-scale fermentation of this strain.The crude metabolites of DL-106 strain were extracted and purified by using decompression silica gel column chromatography,ODS column chromatography and HPLC.Six compounds(4-60-1,6-70-3,7-40-1,7-40-3,6-70-1,7-40-2)were obtained from strain DL-106.The structures of the six compounds were identified as 1,6-phenazinediol,4’,7-dimethoxylisoflavone,2-acetamidophenol,2-aminophenoxazin-3-one,6-Methoxy-1-phenazinol and 1-Methoxyphenazine based on the results of nuclear magnetic resonance and mass spectrometry.When the test concentration was 250 μg/disc,compounds 1-6 could significantly inhibit the violacein production of CV12742 with the inhibition zones of 27 mm,26 mm,17 mm,21 mm,19 mm and 18 mm in diameter respectively.The pigment inhibition ability of compound 3 belonged to the antibacterial activity against the reporter strain,while the pigment inhibition of other 5 compounds belonged to the anti-quorum sensing activity.