Screen For The Antibacterial And Anti-quorum Sensing Activity Of Marine Actinomycetes From Mangrove Sediment In South China Sea And Primary Investigation Of Secondary Metabolites

The inhibitory effect of antibiotics on microbial growth makes it widely used in the medical field,but the problem of bacterial drug resistance accompanying the abuse of antibiotics has become a serious problem that people have to face.While we are looking for new antibiotics,some studies have shown that quorum sensing inhibitors can also control the pathogenic effect of bacteria through the quorum sensing system that mediates bacteria,and will not cause resistance mutations.The main purpose of this study is to find new natural antibiotics or quorum sensing inhibitors from the natural products of mangrove sediment microorganisms in the South China Sea,which is of great significance to solve the problem of microbial drug resistance.In this experiment,10 strains of actinomycetes from mangrove sediment samples in the South China Sea were used as experimental strains,and the indicated strains were Chromobacillus violaceum ATCC 12472,Enterococcus faecalis ATCC 51299,Enterococcus faecium ATCC 35667,Bacillus subtilis ATCC 19659 and Staphylococcus aureus ATCC 33591,ATCC 25923 and ATCC 29213.A strain HN1131 with strong antibacterial activity and quorum sensing inhibitory activity was screened by disc diffusion assay,pigment inhibition assay and biofilm inhibition assay,and its 16S rDNA gene was amplified and sequenced.Based on the morphological characteristics and 16S rDNA sequences analysis,this strain was identified as Streptomyces tanashiensis HN1-131.HN1-131 was optimized under three conditions:culture duration(3 d,5 d,7 d,9 d,11 d,14 d),pH(4,5.5,7,8.5,10)and culture medium(Gauze's medium No.1,A1,N4 medium,AM6-1 medium,YMS medium and optimized Gauze's medium).Based on the cell growth,metabolites production and activity of crude extracts obtained from different culture condition,14 d,8.5 and Gauze's medium No.1 was the best culture condition.The crude extracts of different culture media with quite different activity were analyzed by TLC and HPLC,and the possible active components peaks in HPLC spectrum were preliminarily determined.HN1-131 was fermented on a large scale and its metablites were extracted.The crude extract was separated and purified by reduce pressure silica gel column chromatography,reduce pressure ODS column chromatography and HPLC.Seven compounds E70-1A,E70-9A,E70-9B,E80DA,E80DB,F6A and F6B and a mixture F10 containing two compounds were isolated from the crude extract of the strain.The activities of these seven components were tested.E80DA,E80DB and F10 had good inhibitory activities against CV12472 and SA4;F6B had good antibacterial activity against SA4,but had no inhibitory activity against CV12472;F6A has weak activity against CV12472 and SA4.Finally,the structures of six monomer compounds were identified.Compound E70-1A was dibutyl phthalate,and compounds E70-9A and E70-9B were analogs of E70-1A;Compound F6A is presumed to be a pentapeptide containing proline,threonine,valine,sarcosine and N-methylvaline;Compounds E80DA,E80DB and F6B are presumed to be actinomycin.