Study On Diversity Of Symbiotic Microbes And Antimicrobial Active Components Of Actinomycetes From The Gut Of Blaps Rynchopetera

Objective:The study aimed to comprehensively analyse the diversity of gut microbes in Blaps rynchopetera,and to isolate antimicrobial active compounds from the secondary metabolites of intestinal actinomycetes.Methods:1.High-throughput sequencing was used to analyse the diversity of gut symbiotic microbes from Blaps rynchopetera.2.Bacteria,actinomycetes and fungi were isolated from the gut of B.rynchopetera by using dilution coating and selective culture methods.Methieillin-resistant Staphylococus aureus,Staphylococcus aureus,Escherichia coli,Enterococcus faecalis,Salmonella typhimurium,Pseudomonas aeruginosa,Aspergillus niger,Penicillium expansum and Canidia albicans were selected as indicator strains.The antimicrobial activities of gut actinomycetes against pathogenic microbes were tested by Oxford cup method,and the strains with significant antimicrobial activities were identified by 16 S r RNA sequencing.3.Streptomyces sp.BPA49 and Streptomyces sp.BPA71 were selected for largescale solid fermentation.The extracts with antimicrobial activities were determined by activity tracking method and were separated and purified by silica gel column chromatography and sephadex LH-20 gel column chromatography.The structures of the compounds were analyzed by NMR spectroscopy.The minimum inhibitory concentrations（MICs）of the main compounds were detected by microbroth dilution method to evaluate antimicrobial activities.And the antitumor activities of the main compounds against five tumor cell lines were evaluated by 3-（4,5-dimethylthiazol-2-yl）-5（3-carboxymethoxy-phenyl）-2-（4-sulfopheny）-2H-tetrazolium,inner salt（MTS）method.Results:1.Based on high-throughput sequencing results,Actinobacteria,Firmicutes,Proteobacteria,Bacteroidetes,Ascomycota and Basidiomycota were the dominant microbial communities.Functional analysis showed that gut bacteria played important roles in the growth,development and the defense against pathogenic invasion of the host.The gut fungi were mainly saprophytic fungi and mixed trophic fungi except for those not clearly classified.2.A total of 176 strains of actinomycetes,108 strains of fungi and 103 strains of bacteria were isolated from the gut of B.rynchopetera.The antimicrobial test showed that46 of 176 actinomycetes had different degrees of antimicrobial activities against 9pathogenic microbes,and 18 strains had significant antimicrobial activities.According to the results of 16 S r RNA sequencing and BLAST,the 18 actinomycetes were identified as Streptomyces sp..Among them,two strains of Streptomyces sp.BPA49 and Streptomyces sp.BPA71 showed broad-spectrum antimicrobial activities.3.Five compounds were obtained from the ethyl acetate extracts of secondary metabolites of Streptomyces sp.BPA49 and BPA71,which were methyl 2-furoate（1）,pyrroloformamide A（2）,pyrroloformamide B（3）,methyl 2-（1H-indol-3-yl）acetate（4）and holomycin（5）.Activity test results showed that both Compound 2 and compound 5had broad-spectrum antimicrobial activities.In addition,Compound 2 and compound 5also showed strong cytotoxicity against five tumor cells,including Hela,A549,SMMC-7721,MDA-MB-231 and SW480 cells.Conclusion:Through high-throughput sequencing and fermentation culture technology,the composition of the gut microbes of B.rynchopetera was analyzed,and abundant culturable microbial materials were obtained.Two strains with broad-spectrum antimicrobial activities were selected to study the active ingredients,and five compounds were obtained.Holomycin（5）and pyrroloformamide A（2）were the main antimicrobial components of Streptomyces sp.BPA49 and BPA71,respectively.In addition,compound2 also showed significant anti-tumor activities against five tumor cells.