Isolation And Identification Of Antibiotic-producing Actinobacteria From Lakes In Tibet

Actinobacteria are widely distributed in lake habitats.Many reports suggest that antimicrobial lead compounds are still waiting to be discovered from these natural sources.This paper will establish an integrated approach to exploit antimicrobial secondary metabolites produced by actinobacteria from lakes in Tibet.The results are laid out as following:A total of 158 actinobacteria strains belonging to 19 generas and 90 species were isolated from 22 lake sediment samples,and the dominant genus was Streptomyces.There were significant difference in the biodiversity of actinobacteria from different lake sediment samples.Among them,the largest number of actinobacteria were isolated from sediment samples of Lu-bu-cuo lake.Moreover,6 strains were identified tentatively as novel species by 16S rDNA sequences analysis and phylogenetic tree construction.To establish of pathogen-infected Caenorhabditis elegans in vivo screening model,four factors including the liquid medium,the infection time,the bacterial concentration and the treatment time were investigated.The optimized conditions for Caenorhabditis elegans-Staphylococcus aureus screening model were as follows:20%BHI-M9 as liquid medium,the infection duration was 10 h,the concentration of Staphylococcus aureus was 5×10⁷ CFU/mL,and the treatment time was 24 h.The optimized conditions for Caenorhabditis elegans-Pseudomonas aeruginosa screening model were as follows:20%BHI-M9 as liquid culture medium,the infection duration was 8 h,the concentration of Pseudomonas aeruginosa was5×10⁶ CFU/mL,and the treatment time was 24 h.158 ethyl acetate extracts of fermentation broth were prepared and their antimicrobial activities were determined by using in vitro agar diffusion method and in vivo pathogen-infected Caenorhabditis elegans model.It was found that 64 extracts had antimicrobial activity against at least one of five tested pathogenic bacteria in vitro and 9 extracts had anti-Staphylococcus aureus and/or anti-Pseudomonas aeruginosa in vivo.Furthermore,6 extracts without any in vitro antimicrobial activities had antibacterial activities in vivo.An actinobacterial strain designated as PF160 isolated from a sediment of Lu-bu-cuo lake was identified tentatively as one novel species of the genus Streptomyces by polyphasic taxonomy.The secondary metabolites of the strain PF160 had anti-Pseudomonas aeruginosa activity in vivo.9.6 mg active compound designated as PF160-C1 was separated and purified from 14 L fermentation broth by ethyl acetate extraction,C₁₈ column chromatography,and preparative HPLC.The chemical structure of compound PF160-C1 was elucidatd as 1,6-dihydroxy-8-propylanthraquinone on the basis of extensive spectroscopic analysis including UV,IR,MS,1D-and 2D-NMR experiments.This is the first report that known compound 1,6-dihydroxy-8-propylanthraquinone could be produced by actinobacteria from lake and had antibacterial activity.This work illustrated the potential of actinobacteria from lakes in Tibet as a unique source for antimicrobial secondary metabolites.Moreover,the application of pathogen-infected C.elegans model is valuable for screening of lead compounds with in vivo activity and low toxicity.