Exploration Of Bacterial Resources In The Faeces And The Habitat Soil Of Rhinovithecus Bieti

Yunnan snub-nosed monkeys(Rhinopithecus bieti)are the only nonhuman primates that live near the snow line at high altitude and eat high-fiber foods such as Usnea diffracta Vain.The unique habitats and diets may have nurtured peculiar intestinal microbes and novel natural products in R.bieti,which is a treasure to be explored and utilized by us.In this work,the microflora of the intestinal tract of wild R.bieti and its habitat soil were studied by the method of pure culturing and non-culturing.The bacterial diversity of the microflora in the wild R.bieti intestinal tract and its habitat soil were studied,the antibacterial activity of their fermentation products was tested,various types of functional genes for synthesizing secondary metabolites were screened,and the potential of strains to synthese novel natural products was evaluated.Combined with traditional polyphasic classification methods and genomic analysis,the classification status of potential new taxa was determined.By using the methods of pure culturing,412 strains of intestinal bacteria of R.bieti were isolated and identified,including 221 strains of actinomycetes and 191strains of bacteria.Actinomycetes were distributed in 8 orders,14 families and 25genera of actinomycetes,and bacteria were distributed in 4 phyla,7 classes,10 orders,19 families and 30 genera.Meanwhile,271 strains of soil bacteria were isolated and identified from the R.bieti habitat,including 226 strains of actinomycetes and 45strains of bacteria.Actinomycetes were distributed in 10 orders,14 families and 29genera of actinomycetes,and bacteria were distributed in 3 phyla,5 classes,9 orders,19 families and 22 genera.Among them,75 potential new taxa were obtained,including 41 potential new taxa of actinomycetes and 34 potential new taxa of bacteria,including one potential new genus.Through polyphasic classification and genomic analysis,strain WQ 117^T was identified as a new species of Faecalibacter,named Faecalibacter rhinopitheci WQ 117^T,and strain WQ 047^T was identified as a new species of Sphingobacterium,named Sphingobacterium rhinopitheci WQ 047^T.The results showed that the intestinal tract and habitat soil of wild Yunnan snub-nosed monkeys are rich in bacteria and actinomycetes resources and rich in new species.Through metagenomic sequencing,a total of 112 phyla and 77 classes in kingdom of bacteria were identified in the fecal samples of R.bieti,including 6 classes,21orders,42 families and 115 genera of actinomycetes.The remaining bacteria belong to71 classes,135 orders,302 families and 1172 genera.A total of 45 phyla and 56 classes in kingdom of bacteria were identified in soil samples,including 4 classes,15 orders,23 families and 47 genera of actinomycetes,and other bacteria,52 classes,84 orders,150 families and 354 genera.The species abundance and number of bacteria and actinomycetes identified by metagenome were much larger than those by pure culturing,and the species and numbers of intestinal bacteria of R.bieti were much larger than those of their habitats,indicating that intestinal bacteria are important habitats for exploring microbial resources and their metabolites.The resistance genes in the samples were analyzed.The results revealed that there were 123 kinds of resistance genes in fecal samples of R.bieti and 11 kinds of resistance genes in soil samples.There were more kinds of resistance genes in Yunnan snub-nosed monkey fecal samples,but the abundance of resistance genes was smaller.The species of resistance genes in soil samples were fewer,but the gene abundance was higher.Combined with the species abundance data,this suggested that the horizontal transfer of drug resistance genes among intestinal bacteria in R.bieti was less frequent than that among soil strains,and the number of strains with drug resistance genes in R.bieti intestinal bacteria was aslo less than that of soil strains.The potential of microflora of R.bieti fecal samples to synthesize secondary metabolites was analyzed by anti SMASH.It was found that there were 6 different classes and 40 different kinds of secondary metabolites.This showed that the intestinal microorganisms of R.bieti have the potential for discovering secondary metabolites.Specific PCR primers targeting 12 key genes for synthesizing bioactive compounds were designed,and 285 strains of intestinal bacteria and 267 strains of soil bacteria were screened by PCR amplification to illustrate the potential of these strains to synthesize these natural products or their analogs.The results showed that 76.49%of the intestinal bacteria were positive,while 89.51%of the soil bacteria were positive.The antibacterial activity of the fermenation products of 350 strains of intestinal bacteria and 254 strains of soil bacteria was tested.The results showed that44%of the fermentation products from the intestinal bacteria were positive,while38.58%of that from the soil bacteria were positive.Based on the results of screening for functional genes and measuring of antibacterial activity,as well as macrogenomic prediction,21 strains with the potential of producing new bioactive natural products were selected from the pure culture of fecal samples.The above results showed that the ability of intestinal bacteria of wild R.bieti to generate bioactive natural products were comparable with that of the microorganisms in their habitat.The results of this work showed that the intestinal tract and habitat soil of the wild R.bieti is rich in bacterial resources and have the potential for synthesizing bioactive substances,which provides theoretical and data support for the further exploration of intestinal bacterial resources in R.bieti.