Actinomycetes And Its Secondary Metabolites Associated With Anthill Of Formica Cunicularia

Antibiotic resistance has been a challenge to puzzle the healthcare sector. In paeticular, multidrug resistance is now widespread in pathogens what we are familiar with, such as Staphylococcus aureus and Mycobacterium tuberculosis, even in emerging pathogens such as Acinetobacter baumannii. Ever-increasing antibiotic resistance continues to compromise therapeutic efficacy of the medicines that are currently in use. Thus, there is an urgent need to find new antibiotics and new therapeutic strategies to face this challenge. In fact, the majority of antibiotics in clinical use originated from natural products, mostly from environmental actinomycetes, high rediscovery rates, among other factors, have diminished the enthusiasm for continued exploration of this historically important source. Several well-studied insect agricultural systems have bacterial symbionts that have evolved to produce small molecules that suppress environmental pathogens. These molecules represent an underexplored reservoir of potentially useful antibiotics. The special environmental microbes as well as new strains is one of the important ways to find novel skeleton active nature products。The symbiotic insect-actinomycetes are the special environmental microbes yet to be explored.1. Isolation of anthill soil actinomycetesSoil samples were collected from the anthills in Shandong university (Weihai). 3 kinds of isolation medium were used to separate 15 species of actinomycetes. Molecular phylogenetic study using 16S rRNA gene sequences showed that 15 isolates from anthill soil samples were distributed in 3 genera in Actinobacteria phylogenetic group. These three were Arthrobacter, Lechevalieria and Streptomyces, and the most important of species is Streptomyces containing 13 types.2. Screening of antagonistic strainsThe Gram positive pathogens (Staphylococcus aureus, Bacillus subtilis) and Gram negative pathogen (Escherichia coif) from the isolation and identification of 38 species of actinomycetes were selected as target bacteria to screen bacteria with antibacterial activity. Finally, it is found that 6 species of actinomycetes have antimicrobial effects on S. aureus and B. subtilis and inhibitory effects on S. aureus are stronger than effects on B. subtilis. While none of the strains had antibacterial effects on E. coli. Meanwhile, the metabolites of all the isolated Actinomycetes strains were studied by HPLC, HPLC-MS and antibacterial activity. The results showed that the metabolites of strain A1302 had a wide variety of compounds and also had a strong inhibitory effect on B. subtilis. The secondary metabolites of strain A1314 were demonstrated to have active compounds by HPLC-MS, and also had antibacterial effects on B. subtilis. Finally, the strains A1302 and A1314 were fermented for further study.3. Research on the secondary metabolites of strains A1302 and A1314 as well as the antibacterial activity of isolated compoundsRepeated column chromatography on prep-TLC, silica gel, Rp-18, Sephadex LH-20 and recrystallization were used to separate the secondary metabolites of strain A1302 and A1314, and more than 20 compounds were isolated. Elucidation of the structures was performed by IR, UV, ESI-MS, HR-ESI-MS, 1D NMR and 2D NMR. A total of 10 coupounds were separated and identified, with 2 of them were found as new compounds.A1302 strain was cultured on modified Gause’s synthetic liquid medium and 7 compounds were isolated and determined as two new phenoxazinone alkaloids, viridobrunnines A and B, along with two known analogues exfoliazone and chandrananimycin D, one macrolide antibiotics curvularin, N-(2-hydroxy-5-methylphenyl)acetamide and stigmasterol. The antibacterial activity of 7 isolated compounds was assayed. Viridobrunnine B exhibited potent antibacterial activity against B. subtilis with inhibition zone from 13 mm to 15 mm and viridobrunnine A and exfoliazone had weak inhibition. Chandrananimycin D and curvularin exhibited moderate inhibitory effect on S. aureus.A1314 strain was fermented with modified Gause’s synthetic liquid medium and the fermentation broth was extracted with EtOAc. A total of 3 compounds were isolated and identified including a known rearranged zizaane-type sesquiterpene strepsesquitriol, griseoluteic acid and N-(2-hydroxy)acetamide. The antimicrobial activity was evaluated. Griseoluteic acid exhibited inhibitory effect on Gram positive and Gram negative pathogens and had a stronger antibacterial activity against Gram positive pathogens. Griseoluteic acid had a moderate antimicrobial effect on S. aureus and B. subtilis.