Classification And Genomic Analysis Of Two New Species From The Feces Of Tibetan Antelope In Qinghai-Tibet Plateau

ObjectiveMany wild animals are hosts for important human infectious disease pathogens,especially wild animals that live in special environmental and climatic conditions such as plateaus and tropics,and may carry and spread some known and unknown pathogens.Some of these pathogens have potential to cause new infectious diseases.Studies have shown that the feces of Tibetan antelope in the Qinghai-Tibet Plateau contain many new species and uncultured bacterial groups.This study used the strategy of culturomics to isolate and identify the new species contained in Tibetan antelope fecal specimens.This will help us to broaden the research on the diversity of wild animal feces?intestines?on the Qinghai-Tibet Plateau,and provide a scientific basis for coping with,preventing and controlling new infectious diseases that may occur in the future.Methods?1?Using the method of Culturomics,72 culture schemes were designed by multiple combinations,and 104 Tibetan antelope feces collected in the Hoh Xil Nature Reserve in Qinghai Province from May 13 to 15,2014 were isolated and cultured.According to the colony morphological diversity?colony color,size,texture,marginal condition,etc.?,single colony picking,purification,the amplification and sequencing of 16S rRNA gene sequence were performed.The sequence of the 16S rRNA gene fragments of the strains was blasted by GeneBank and EzTaxon database to complete the preliminary identification.Based on the above results,the preservation of the strains was carried out in accordance with the strains diversity.?2?Based on the preliminary identification results of the strains,the strains were analyzed for microbial diversity.?3?For the potential new species of bacteria,the taxonomic identification and genomic analysis should be completed,including:phenotypic,physiological and biochemical and cell wall chemical classification characteristics experiments,phylogenetic analysis,virulence genes and other genomic analysis and discovery of biological significance.Finally,the nomenclature of new species and the deposition of type strains in CGMCC?China General Microbiological Culture Collection Center?and DSMZ?Deutsche Sammlung von Mikroorganismen und Zellkulturen?was done.Results?1?In this study,976 strains had been isolated from the feces of Tibetan antelopes at the Hoh Xil Nature Reserve,Qinghai-Tibet Plateau,China,and were identified into 3 Phylum,5 Class,16 Order,30 Family,60 Genus,117Species successfully.The dominant genus is Arthrobacter,Paenibacillus,Enterococcus and Bacillus.Among them,67 strains are potential new species,which can be affiliated to 21 species and 14 genera.And 28 strains are possible medically significant strains,belonging to 6 species and 4 genera,including Streptococcus,Mycobacterium,Rhodococcus and Roseomonas.Among the new suspected bacterial species,the four strains of potential new species?78^T,601,X-1125^T and X-1174?have completed the identification and nomenclature of new species.?2?Based on evidence collected from the phenotypic,genotypic and phylogenetic analyses,we proposed a novel species named Nocardioides houyundeii sp.nov.The type strain is 78^T?=CGMCC 4.7461^T=DSM 106424^T?.Two novel Gram-stain-positive,irregular rod-shaped bacterial strains,78^T and601,were cream in color,circular,smooth and convex when cultured on brain heart infusion agar supplemented with 5%sheep blood.Phylogenetic analysis based on the16S rRNA sequences revealed that strains 78^T and 601 belongs to the genus Nocardioides,sharing the highest similarity with Nocardioides solisilvae JCM 31492^T?98.3%?,Nocardioides gilvus XZ17^T?97.4%?and Nocardioides daejeonensis JCM 16922^T?97.4%?,while the 16S rRNA genes of strains 78^T and 601 were highly similar?99.7%?to each other.The average nucleotide identity values between strains 78^T and 601 was 98.9%?well above the species threshold?,but their values were lower than the 95–96%threshold with the three closely related type strains.The DNA G+C content of strains 78^T and 601 were 71.2 and 71.3 mol%respectively.MK-8?H₄?was the predominant respiratory quinone and ll-2,6-diaminopimelic acid was the diagnostic diamino acid in its cell wall peptidoglycan.Its polar lipids contained diphosphatidylglycerol,phosphatidylglycerol,phosphatidylinositol,an unidentified phospholipid and an unidentified lipid.The major fatty acids?>10%?were C_17:1?8c,iso-C_16:0 and C_18:1?9c.?3?Based on the presented data,strains X-1125^T and X-1174 hereby represent a novel species of the genus Paraliobacillus,for which the name Paraliobacillus zengyii sp.nov.is proposed.The type strain is X-1125^T?=DSM107811^T=CGMCC 1.16464^T?.Two rod-shaped,slightly halophilic and extremely halotolerant bacterial strains?X-1125^T and X-1174?were Gram-stain-positive,facultatively anaerobic and motile with peritrichous flagella.Based on results of 16S rRNA gene sequences,phylogenetic and phylogenomic analyses,their nearest phylogenetic neighbors were Paraliobacillus sediminis KCTC 33762^T?98.4%?,Paraliobacillus quinghaiensis CGMCC 1.6333^T?96.9%?and Paraliobacillus ryukyuensis NBRC 100001^T?95.9%?while the 16S rRNA genes of strains X-1125^T and X-1174 were highly similar?99.7%?to each other.In silico DNA–DNA hybridization value between strains X-1125^T and X-1174 was 97.8%?well above the species threshold?,but their values were lower than the 70%threshold with the three closely related type strains.Strains X-1125^T and X-1174 had DNA G+C contents?mol%?of35.2%and35.1%,respectively.Thepolarlipidscomprised diphosphatidylglycerol,two unidentified phospholipids and four unidentified lipids.MK-7 was the sole menaquinone.The cell wall contained alanine,glycine,glutamic acid and meso-diaminopimelic acid.The major fatty acids?>9%?were anteiso-C_15:0,anteiso-C_17:0 and C_16:1?11c.?4?The chromosomes of strains 78^T and X-1125^T were 3,840,802 bp in length and 3,728,451 bp in length and to possess 3,437 and 3,470 coding sequences?CDS?,respectively.By functional clustering analysis with COG and KEGG databases,most of these genes are related to the metabolism?including the transport and metabolism of amino acids,carbohydrates,nucleotides and lipids,etc.?.The results of the 78^T and X-1125^T NR database annotations supported the classification of genus Nocardioides and Paraliobacillus.Based on the slightly halophilic and extremely halotolerant nature of genus Paraliobacillus,we further compared their genomic composition.Multiple resistance and pH gene clusters?mrp gene cluster?were found,encoding a Na⁺/H⁺antiporter and might play a major role in sodium tolerance and pH homeostasis.In addition,by comparing with PHI and VFDB databases,12 genes associated with two-component systems of Mycobacterium tuberculosis,which is essential for the virulence and growth of bacteria,and 25 virulence-related genes were found in genome of strain 78^T.24 virulence-related genes were also found in genome of X-1125^T.The function of the related virulence factor and the pathogenicity of this strain remains to be further studied.Conclusion?1?In this study,976 strains had been isolated from the feces of Tibetan antelopes at the Hoh Xil Nature Reserve,Qinghai-Tibet Plateau,China,and were identified into 3 Phyla,5 Classes,16 Order,30 Family,60 Genera,117Species successfully.There are 57 strains of suspected new species,of which 28strains are possible medically significant strains.?2?Strains 78^T and 601 were representatives of a novel species of genus Nocardioides,for which the name Nocardioides houyundeii sp.nov.is proposed.The type strain is 78^T?=CGMCC 4.7461^T=DSM 106424^T?.Strains X-1125^T and X-1174 represent a novel species of the genus Paraliobacillus,for which the name Paraliobacillus zengyii sp.nov.is proposed.The type strain is X-1125^T?=DSM 107811^T=CGMCC 1.16464^T?.The genomic analysis of strains 78^T and X-1125^T revealed that there are multiple genes related to host interaction and virulence-related genes,and the related virulence factors and pathogenicity of the strain remain to be further studied.