The Primary Study On The Secondary Metabolites Of A Streptomyces Globisporus Strain WA5-2-37 With Anti-MRSA Activity Isolated From Cockroach Intestinal Tract

Methicillin-resistant Staphylococcus aureus?MRSA?is known as"Super-bacteria"because of its resistance to common antibiotics in clinic.At present,the infection rate of MRSA has surpassed that of hepatitis B and AIDS,and become the most incurable infectious diseases all over the world.Therefore,it is urgent to develop new,safe and effective anti-MRSA drugs.Insect intestinal endogenous microorganisms are closely related to the host's physiological functions such as nutrient uptake,growth and development,and resistance to pathogen invasion.Unlike other microorganisms from soil or plant,the metabolic pathways of insect intestinal endogenous microorganisms may be activated and regulated by the intestinal environment so that it can produce many distinctive secondary metabolites.Therefore,the secondary metabolites of insect intestinal endogenous microorganisms may have a potential to discover the new natural products with great bioactivity.We have isolated 18 strains with anti-MRSA activities from the intestinal tract of cockroach in early study.In this research,the MRSA was used as indicator and the strain WA5-2-37 with great anti-MRSA activity was screened out from 18 strains.Then we investigated species characterization of the strain WA5-2-37 by means of morphological identification,molecular biological identification and phylogenetic tree analysis.Moreover,we used single factor optimization and response surface methodology to optimize its fermentation,and studied the secondary metabolites with anti-MRSA activity preliminarily by bioassay-guided and chemical separation methods.The main results of this study are as follows:1.Screening of the strains with great anti-MRSA activity:The strain WA5-2-37 with great anti-MRSA activity was screened out by means of agar or filter diffusion method which was used the MRSA as indicator,and its inhibition zoom of was 22.30±0.42 mm.2.The identification and characterization of strain WA5-2-37:The strain WA5-2-37 was tentatively identified as Streptomyces globisporus based on the results of the morphological characteristics on the medium,gram staining,scanning electron microscope observation,16S rDNA sequence alignment and phylogenetic tree analysis.The accession number of the 16S rDNA sequence in GenBank was MH304280.3.Optimization the fermentation of strain WA5-2-37:We used the single factor optimization and response surface methodology to optimize the fermentation of strain WA5-2-37.According to the results,we concluded that optimum conditions of fermentation were as follows:the seed age was34 h,the fermentation time was 7 d,the initial pH of the medium was 6.9,liquid filling quantity was 300 mL/500 mL,the concentration of inoculation was 7%,the temperature of fermentation was 29.8 centigrade and the speed of shaker was 160 rpm/min.Under the optimum conditions,the anti-MRSA activity of the secondary metabolites was the best and the its inhibition zone was 23.82±0.21 mm.4.Isolation and characterization of the secondary metabolites with anti-MRSA activityThe strain WA5-2-37 was extended fermentation according to the optimum conditions.Then the multiple chromatographic approaches including TLC,silica gel,Sephadex LH-20 column chromatography,HPLC and Semi-preparative HPLC,etc.,and bioassay-guided were performed for the isolation of the secondary metabolites with anti-MRSA activity.Eight compounds were isolated from the ethyl acetate fraction,and identified by means of spectroscopic methods such as NMR,MS,UV,etc.The compounds in this study were identified as dibutylphthalate?Compound 1?,Actinomycin X₂?Compound 2?,?6E?-4-methoxy-5-?methylsulfone?-6-?nitroethylene?-1,6-dihydro-2'-bipyridine?Compound 3?,7-methoxy-5-?2-pyridyl?-isothiazole-[4,5-b]-pyridine?Compound 4?,3-aminodemethyl-?oxy?-carbamide?Compound 5?,Cyclo-?Alanine-Proline??Compound 6?,Cyclo-?Valine-Proline??Compound 7?and Cyclic-?L-Proline-L-Tyrosine??Compound 8?.5.Antibacterial and antifungal activity evaluation of the secondary metabolites:The activities of these compounds against the growth of MRSA,Staphylococcus aureus,Escherichia coli and Candida albicans were evaluated.The results indicated that compounds 2-3 exhibited great antibacterial and antifungal activity against the MRSA,Staphylococcus aureus,Escherichia coli and Candida albicans.Other compounds displayed antibacterial and antifungal activity in different degrees.