Isolation And Selection Of Antagonistic Streptomyces From The Soils In Extreme Environmental And Its Metabolites

Streptomyces is a wide spread genus in natural soils, in which the species are filamentous and gram-positive bacteria, and most of these microbes can produce abundant intermediate metabolites, such as enzymes, antitumor agents, immune inhibitors, vitamins and antibiotics etc. It is well known that most of antibiotics used in the clinic were produced by Streptomyces, including actinomycin, erythromycin, arloammonia cephalosporins, and nicktoxin etc. On the other hands, the increasing clinical utilization of antibiotics causes a serious antibiotic resistance of pathogenic bacteria. Nowadays, we urgently need to develop new antibiotics to copy with antibiotic resistance of bacteria. Isolation of Streptomyces from extreme environment or some unexplored regions is an effective way to obtain novel antibiotics and bioactive substances. In this study, we collected the soil samples from the rhizosphere and bulk in the desert of Hexi Corridor and from the Zha jia zang bu, Ge, er-mu, Na cuo hu, and Wu li in the Tibet Plateau. From these soils we isolated Streptomyces strains, and identified those using 16 S rRNA gene sequence analysis. Furthermore, antimicrobial activities of these Streptomyces strains were tested against the indicator microbes. We also carried out the solid-state fermentation of strain 5-1-3 and separated its antibacterial metabolites from the fermentation media. The results have been achieved are as follows.A total of 164 Streptomyces strains were isolated from the soil samples. Among them, 113 strains were isolated from rhizosphere soils in Hexi Corridor, 30 strains were isolated from bulk soil in Hexi Corridor. These strains contain albosporus, cinereus, griseorubroviolaceus, pink spore groups, ball spore groups, yellow groups, Hygroscopicus groups, green groups, and lilac grey groups. The majority of strains in rhizosphere soils are albosporus, lilac grey groups, yellow groups, and Griseorubroviolaceus, and the strains from bulk soil are albosporus and yellow groups. And 21 strains Streptomyces seperared from the soil of the Tibet Plateau used the method of E. coli BTH 101 antigbacteria screening, and the major strains are yellow groups, griseorubroviolaceus, and green groups. The majority of strains in the soils from Tibet Plateau can produce soluble pigment in different colors.The molecular identification of strains was performed. A total of 52 strains of Streptomyces were identified using 16 S rRNA gene sequencing, and then the 16 S rRNA gene sequences were blasted agaist the NCBI database. The results showed that the 50 strains have high sequences similarity(99~100%) with at least one Streptomyces strain in the database. The results also showed that there are abundant Streptomyces resources in the rhizosphere soil and bulk soil from in Hexi Corridor, and the dominant and widespread strains are S. coerulescens, S. variabilis and S. coeruleofuscus.By evaluating antimicrobial activities of isolated Streptomyces we selected 134 strains having antibacterial activity and 33 strains with antifungal activity. Among those isolated from the Hexi Corridor, 98 strains could inhibit the growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, only 8 strains could inhibit E.coli BTH 101; a total of 38 strains could inhibit the growth of Phytophthora infestans, cabbage wilt bacteria(F. oxysporum sp. conglutinans) and Botrytis cinerea. A total of 21 strains isolated from the Tibet Plateau could inhibit the growth of E.coli BTH 101.The strains having resistant to E.coli BTH 101 were further assessed using indicator bacteria methicillin resistant staphylococcus aureus(MRSA). Two strains, 5-1-3 and EJZB3-1, which have a strong antagonistic effect on MRSA have been successfully selected. Therefore, we carried out the solid-state fermentation of strain 5-1-3 and the antimicrobial metabolites in the fermentation media were separated, purified and identified by organic solvent extraction, multiple column chromatography and high performance liquid chromatographic methods. Finally, we have obtained a high yield(20 mg/L) with pale yellow compound which has anti-S. aureus, anti-MRSA and anti-tumour activities from strain 5-1-3 fermentation media. Using nuclear magnetic resonance(NMR) analysis, this compound was identified as echinomycin. In addition to echinomycin, there are other substances in the fermentation products of strain 5-1-3, which are further separated and identified. It will be hopefully obtain new bioactive substances in further study.