Study On The Diversity And Biological Activity Of Fecal Actinobacteria

According to the antibiotics screening or research achievements of microbe genomerecently, actinomycetes remain a rich source for natural products screening. Nowadays, newdrugs, especially antibiotics, are urgently needed to counter and reverse the spread ofantibiotic resistant pathogens and to combat life-threatening diseases as well as new diseases.All of these compel the scientists to search some actinomycetes from new sources. Animals'gut is a typical and unique environment, few researches study the fecal actinobacteria assource. In this study, we researched the diversity of fecal actinobacteria usingculture-independent and culture-dependent approaches, and got new species by polyphasictaxonomic research, and further screened and evaluated their bioactivities againstpathogenetic microorganisms and tumor cell and enzymatic activities. One strain with highantitumor activity was further studied on its secondary metabolites. The current study willprovide a new way to exploit and utilize the actinomycetes sources.We studied the diversity of bacteria16S rRNA gene in seven fecal samples collectedfrom Yunnan Safari Park by454sequencing. A total of42540bacteria V3sequences of16SrRNA genes were obtained and clustered into2979(a threshold of100%identity) uniquesequences and876(a threshold of97%identity) OTUs (operational taxonomic units), andthey distributed at Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, Actinobacteria,Spirochaetes, Cyanobacteria, Fibrobacteres, Tenericutes, Acidobacteria, Verrucomicrobia,Deferribacteres and TM7, total13phylums. Amoung them, Firmicutes and Bacteroideteswere dominant in all fecal samples. However,34.7%of the bacterial sequences can not beidentified into any of the known phylum, indicating that these sequences represented somenew species. The total OTUs were ditributed in116genera. Diversity Index curves showedthat the diversity in omnivorous Rhinopithecus roxellanae, Hlobates hoolock, Pan troglodytesand Pavo cristatus samples was high, but was relative low in Ursus thibetanus, Ailuiopodidaemelanoleuca and Panthera tigris altaica samples. The diversity from Primates' fecal sampleswere similar, and Ursidae's fecal samples were similar, too. The difference of the diversitybetween omnivorous Pavo cristatus and carnivorous Panthera tigris altaica was very large.These indicated the bacteria diversity was associated with phylogenetic relationship and feeding habit. Thereinto,3.2%sequences belong to Actinobacteria, including103OTUs. For7samples, the proportion of Actinobacteria in Panthera tigris altaica was the largest,accounting for15%, the novel Actinobacteria was the most, too.Attaining culturable strains is essential for studying and utilizing the microorganisms,thus the isolation method of fecal Actinobacteria was researched. We found that drying andheating up the fecal samples can greatly increase the rate of the actinobacteria. Moreover,many kinds of inhibitors and chemical defined media were suitable for isolation of fecalactinobacteria. Base on this,923purified strains of actinobacteria were isolated from28animal feces samples, and344strains of them were identified as35genera, one of them isnew genus candidate. Streptomyces was the primary filamentous actinobacteria group and upto107colony forming unit (CFU) per gram of dry samples. Rhodococcus was second mostabundant, and this group appeared in2/3samples; The members of suborder Micrococcineaewere main non-filamentous actinobacteria. This study aimed at aerobic actinobacteria, notrefer to anaerobic actinobacteria.The new genus candidate was subjected to polyphasic taxonomy study including analysisof morphological characteristics, physiological and biochemical tests, chemotaxonomicproperties, phylogenetic analysis based on16S rRNA gene sequences. Strain YIM100590was identified as a member of the novel genus of the family Micrococcaceae, the nameEnteractinococcus gen. nov. was proposed. Enteractinococcus coprophilus sp. nov. is the typespecies of new genus.Through screening of bioactivities on the fecal Actinobacteria,76strains showed activityagainst K562cell and71strains showed activity against HL60cell by using MTT assay;26strains showed antimicrobial activity using the agar plate diffusion method; More than90%tested strains were positive for alkaline phosphatase, acid phosphatase, leucine arylamidaseand naphthol-AS-BI-phosphohydrolase activity,103could hydrolyze cellulose and174showed keratin activity; The functional gene screening results showed that34strains hadpolyketide synthase I (PKS I) gene,30strains had PKS II gene,58strains had non-ribosomalpeptide synthetase (NRPS) gene and22strains had cytochrome P450hydroxylase (CYP)gene.Nine compounds were isolated and purified from80liter fermentation broth of strainYIM100525, which had high antitumor activity. The structure of compounds1-9wereconfirmed by spectroscopic analysis, and compounds1-3had novel structures.The results of this work firstly showed that fecal samples harbored huge quantity ofbacteria, and many groups at phylum level were novel. Secondly, fecal actinobacteria wereabundant. The strains of genera Streptomyces and Rhodococcus were dominating, the members of suborder Micrococcineae were also abundant. This is a distinct feature for groupcomposition of fecal actinobacteria. Many novel actinobacteria existed in feces, too. Thirdly,the proportion of bioactive strains was large. We infered fecal actinobacteria was veryimportant from a nutritional, physiological and immune point of view. Further studyingmechanisms of these function of actinobacteria is significant for the health of host. Fourthly,abundant compounds with different activities were obtained from the fecal actinobacteria,which showed fecal actinobacteria was an important resource for seeking active leadcompound. In conclusion, like soil, marine and endophytic actinobacteria, fecal actinobacteriais a significant source for developing drug and other manufacture. Thus, the research,protection and utilization of fecal actinobacteria should be strengthened. This study has layedthe foundations for development and utilization of fecal actinomycete resources.