Discovery And Genomic Analysis Of New Species From The Feces Of Bats

ObjectiveBats are the natural hosts of a variety of pathogens,which can cause numerous emerging infectious diseases.In recent years,people have paid more attention to bats because they can carry various highly pathogenic viruses,however,the research on the bacterial community of bats'feces(intestines)and whether they carry bacterial pathogens is rare.In this study,16S r DNA amplified sequencing technology and combined with the traditional culture and isolation method to analyze the bacterial flora from five fecal samples of different bat species,which were collected from five different geographical locations in China.Enriched the microbial catalogues,and evaluated the potential pathogenicity or public health significance of the newly discovered bacteria,in order to prevent and reduce the occurrence of various infectious diseases,and provide scientific basis for China to formulate the public health strategies for the effective prevention and control of emerging infectious diseases.Methods(1)Using 16S r DNA amplicons sequencing technology for the 16S r DNA high variable region(V3-V4)sequencing,and the sequenced results were compared with the Silva database.When the sequences similarity is more than97%was listed in an operational taxonomic units(OTU)and the highest frequency was chosen as the representative of OTU sequences,the KRONA software was used to make statistics on the classification results.(2)According to the method of Culturomics,a variety of culture schemes were designed to isolate and culture for fecal samples with traditional isolation and culture methods.Individual colonies with different color,size,edge and surface smoothness of bacterial colonies were selected for pure culture and preservation.The nucleic acids of isolated strains were extracted for 16S r RNA gene amplified sequencing to align with the sequences of Gene Bank and Ez Taxon databases.According to the preliminary results,the strains of different types were purified according to the multiplicity principle,and then put into glycerin tubes and stored at the temperature below-40?.(3)According to the preliminary identification results,the known species of bacteria were analyzed for microbial diversity.For the potential new species of bacteria were analyzed by the physiological and biochemical characteristics,including phenotypic,biochemical,bacterial components,phylogenetic relationship,virulence genes and other genomic analysis and discovery of biological significance.Finally,the new species of bacteria were named and the type strains were sent to China General Microbiological Culture Collection Center(CGMCC)and Japan Collection of Microorganisms(JCM)for preservation,and then the certificates of deposition were obtained.Results(1)The analysis results of 16S r DNA amplicons sequencing technology indicated that there was a diversity of bacteria in the fecal samples of bats,with a total of 752 OTUs were generated by clustering,belonging to 19 Phyla,34Classes,68 Orders,116 Families and 241 Genera.Among the five fecal samples,Proteobacteria was widely distributed in the fecal samples of Taphozous spp.,Rhinolophus spp.and Myotis spp.,accounting for 84.63%,61.70%and 51.97%,respectively.The most widely distributed phylum in the fecal samples of Rousettus spp.is Bacteroidetes(52.97%),followed by Proteobacteria(30.55%)and Actinobacteria(7.33%).In the samples of Hipposideros spp.,Bacteroidetes,Proteobacteria and Firmicutes accounting for 45.42%,29.70%and 22.39%,respectively.(2)In this study,195 strains of bacteria were successfully isolated and identified from fecal samples by traditional isolation and culture methods,mainly distributed in 4 Phyla,6 Classes,10 Orders,30 Families,50 Genera and110 Species.The dominant genera are Microbacterium,Enterococcus and Rhodococcus.Among the isolates,there are 93 strains were identified as potential new species,which can be affiliated to 26 species and 14 genera.The genus of Microbacterium is the dominant genus,accounting for 19.4%of the potential new species.There are 19 strains were identified as potential pathogens,accounting for 9.74%of the total number of obtained strains.They can be divided into 15 species,belonging to 8 bacteria genera,among which the genus of Enterococcus and Staphylococcus account for 26.32%and 21.05%of the pathogenic bacteria,respectively.(3)Based on the results of phenotype,physiological and biochemical characteristics,phylogenetic relationships and genomic analysis,it is supported that the strains HY041^T and HY039 can be divided into a new species of genus Apibacter,for which the name Apibacter raozihei sp.nov.,with the type strain is HY041^T(=CGMCC 1.16567^T=JCM 33423^T).Strains HY041^T and HY039are facultatively anaerobic,Gram-stain-negative,rod-shaped,non-flagella and spores.The colonies are round(1-2 mm in diameter),shiny,convex and yellow.The 16S r RNA sequence similarity of HY041^T and HY039 are most closely with A.adventoris wk B301^T(95.2%,95.2%)and A.mensalis R-53146^T(94.0%,93.9%).The ANI values(73.8-74.8%)and DDH values(22.6-26.2%)with their genomes are both lower than the threshold for the demarcation of prokaryotic species boundaries(ANI,95-96%;DDH,70%).The G+C contents of HY041^Tand HY039 are 32.2mol%and 32.0mol%,respectively.MK-6 is the sole menaquinone and polar lipids include phosphatidylethanolamine,glycolipid,two unidentified amino lipids and four unidentified lipids in strain HY041^T.The main fatty acids(>10%)are C_16:0,iso-C_15:0and iso-C_17:03OH.The genome of HY041^Tcomprised 3,293,048 bp with 2,912 genes(Coding genes:2841;r RNA:9;t RNA:43).By comparing with the database of Virulence Factors of Pathogenic Bacteria,it is found that there are some pathogenic genes in the genome of the strain HY041^T,encoding virulence factors which may damage or kill the cells,such as hemolysin(Acp C,cyl G and hyl A)and urease(ure A,ure B,ure F,and ure G).ConclusionThere are hundreds of bacteria in bat feces,including many potential pathogens and unknown bacteria.Apibacter raozihei sp.nov was discovered and analyzed,there are a variety of virulence genes were found in the genome of strain,which may be pathogenic.Its medical significance remains to be further studied.