| Nowadays,due to the excessive dependence on antibiotics in the medical field,most pathogens are insensitive to and tolerant to antibiotics.How to solve the pathogen resistance has become a major problem in the medical research.Usually,pathogenic bacteria cause human infection through the production of virulence factors,and most of the virulence factors are regulated and controlled by the quorum sensing system of the pathogen itself.The substances that have inhibitory effects on the quorum-sensing system of pathogens do not interfere with the nonnal growth of bacteria,but they can directly inhibit pathogenic bacteria from producing virulence factors.They can control the production of pathogenic bacteria virulence factors from the source.Therefore,using anti-group induction active substances to control or synergistically control resistant bacteria has become a research hotspot.The main purpose of this study was to isolate and screen compounds with anti-quorum-sensing activity from the metabolites of marine actinomycetes.In this research,119 marine actinomycetes strains were isolated from the samples in the Lianyungang beach mud by using the plate dilution method.Chromobactierium violaceum,served as a quorum screening reporter strain,was used to measure the anti-quorum sensing activity of the isolated marine actinomycetes by using disc diffusion assay.Three strains with higher activity were screened out from them(101,106 and 1).Among them,No.106 had the most stable anti-quorum sensing activity,which was determined by multiple fermentation cultures.The inhibitory radius of purple pigment production in C violaceum created by the extract of strain 106 was always above 2.0 mm.The intracellular and extracellular distributions of the active substances in the three active strains were studied using the pigment inhibition assay.The results showed that the anti-quorum sensing active substances of No.106 strain were mainly distributed in the spent culture medium.While the bacterial cells had a certain role in promoting the production of the purple pigment;the active substances of No.101 were mainly distributed in the bacterial cells.Based on the 16S rDNA sequence analysis and the morphological characteristics,strain No.101.106 and 1 were identified as Streptomyces koyangensis,Nocardia dassonvillei and Micromonospora yangpuensis,respectively.Because of the best activity,strain No.106,was selected for the optimization of the culture conditions and the medium component.The results showed that the optimum culture conditions of the strain were:rotation speed 140 rpm,fermentation time 14 day,culture temperature 30 ?.Soybean meal was a good nitrogen source for this strain,because it could significantly increase the production of the active metabolites.If only considered the anti-quorum sensing activity of the strain,the best medium component for 106 was to adjust the carbon source to 1%soluble starch,nitrogen source to 0.6%soybean powder and add 3%sea salt on the basis of GAUZE's No.1 medium.In the case that both strain metabolite activity and yield were considered,the best medium components differed from the former only in the absence of sea salt.Using the optimized medium and culture conditions,NO.106 was subjected to large-scale fermentation and crude extraction of metabolites.The extracts were isolated and purified by decompression silica gel column chromatography,ODS column chromatography,Sephadex gel column chromatography and high performance liquid chromatography.Three compounds 106-4-5,106-6-1 and 106-4-3 were isolated from the strain 106.Two compounds,106-4-5 and 106-6-1,were identified as Questiomycin A and its homologues by nuclear magnetic resonance and mass spectrometry.106-4-3 was identified as Benzaldehyde.All three compounds had strong anti-quorum sensing activity.Their inhibition radius of purple pigments production in C.Violaceum were 9.5,4.5 and 4.0 mm at 250 ?g/slice,respectively. |
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