| The study was derived from the thought “animal pathogens and its pathogenic to human”,focused on flora of respiratory specimen of Marmota himalayana in the Qinghai-Tibet Plateau,China.Combining with epidemiological survey and the laboratory analysis,applying PacBio highthroughput sequencing method,microbial culturomics and unknown new pathogen identification technology,the flora distribution was clearly illustrated.Three novel Streptococcus spp.were isolated and Streptococcus marmotae sp.nov.,Streptococcus himalayana sp.nov.and Streptococcus halotolerans sp.nov.were named respectively.According to the results of the study,it not only stated the classification status of the three novel Streptococcus spp.and phylogeny relationships in the Streptococcus genusclearly and accurately,but researched the whole genome characteristics,virulence factors by comparative genomic method.In the study,16 S rRNA amplification,cloning and sequence analysis were used to explore the respiratory tract flora of Marmota himalayana from Qinghai-Tibet Plateau of China.It showed that Streptococcus flora accounting for 49.8%,followed by Escherichia(11.1%)and Veillonella(8.5%)respectively.Obviously,Streptococcus genus is a advantage flora.Furthermore,among the clones of all unknown species,the number of Streptococcus genus clones was 69.1%,suggesting that a great large number of novel Streptococci species were distributed in the respiratory tract flora.It provided strong clues for the discovery of Streptococcus sp.nov.by separating and culturing.According to the clue,we screened 75 respiratory tract tissues samples of Marmota himalayana by microbial culturomics method,and successfully isolated three novel species of Streptococcus spp.Based on biochemical reactions,morphology and phylogenetic analysis revealed that the three novel species represents three new species of the Streptocccus genus.According tothe naming rules of the new international microbes,combined with the characteristics of the isolates,S.marmotae sp.nov.,S.himalayana sp.nov.and S.halotolerans sp.nov.were proposed respectively.The morphological characteristics of three unknown new species were similar,cells stain Gram-positive,are non-spore-forming cocci,0.5-1.0 ?m in diameter and occur in pairs or short chains.Colonies on blood agar are small,raised,and adherent with regular margins,0.5-1.0 mm in diameter and ?-haemolytic at 37 °C for 48 h.Cells are facultatively anaerobic,catalasenegative and non-motile.Growth occurs at 22,30,37 and 42 °C but not at 4or 15 °C.The growth of S.marmotae and S.himalayana occurs in the presence of 2.5% NaCl but not in broth containing 4.5 or 6.5% NaCl.S.halotolerans could grow in the medium with NaCl concentration up to 9.0%.Three novel species were further characterized biochemically using the Rapid ID 32 Strep,API 50 CH and API ZYM systems.Their biochemical characteristics together,such as acid production profile from different sugars and enzymatic activities,placed them as a member of the genus Streptococcus,but did not correspond to any recognized species of the genus Streptococcus.Based on their phenotypic characteristics and in particular the phylogenetic findings(DNA-DNA hybridization,3 phylogenetic trees built16 S rRNA,3 house-keeping genes,and core genes from core genes of 65 Streptococcus whole genomes),it is obvious that S.marmotae form a distinct lineage not affiliated with any known species of the genus Streptococcus but closely related to some described species(S.suis,S.minor and S.ovis),and thus falling into the suis “species group”.S.himalayana forms a distinct subline and exhibits specific phylogenetic affinity with S.marmotae,and thus falling into the suis “species group” too.S.halotolerans forms a distinct subline and exhibits specific phylogenetic affinity with S.thoraltensis and S.hyovaginalis falling into the mutans “species group”.Digital DNA–DNA hybridization studies showed less than 70% DNA–DNA relatedness between three unknown new species and other species of the genus Streptococcusdeposited in GenBank database,which firmLy supports that three unknown new species belonged to Streptococcus genus and formed three new Streptococcus species respectively.The complete genome of the type strains of the three novel species has been sequenced by PacBio high-throughput sequencing technology and analysis.The circular chromosome of S.marmotae HTS5 T comprises2,322,791 bp with an overall G + C content of 41.6 mol%.The genome encompasses 2,377 CDSs,58 tRNAs,and 12 rRNAs,with 2 plasmids(one size is 3,762 bp and the other is 5,997 bp).It also contains 11 genomic islands.The circular chromosome of S.himalayana HTS2 T comprises 2,275,471 bp with an overall G+C content of 41.3 mol%.The genome encompasses 2,195 CDSs,57 tRNAs,and 12 rRNAs.It also contains 6 genomic islands.The circular chromosome of S.halotolerans HTS9 T comprises 1,823,556 bp with an overall G + C content of 39.9mol%.The genome encompasses 2,068 CDSs,68 t RNAs,and 18 rRNAs.It also contains 9 genomic islands.By comparision with VFDB database,we found three novel species carry putative virulence genes for the following factors: Has,ClpC and ClpE were the common virulence factors of S.marmotae HTS5 T,S.himalayana HTS2 T and S.halotolerans HTS9 T.PsaA and PavA,adherence factors associated with pneumococcal infections were detected from genomes of S.marmotae HTS5 T and S.himalayana HTS2 T.Their capability to cause human and/or animal disease will be studied in the future.By compareing genome sequence of three novel Streptococcus species with the virulence genes of VFDB database by BLAST analysis,it showed that Has,ClpC and ClpE were new virulence factor of three novel Streptococcus species.As the main aggressive surface adhesion of Streptococcus pneumoniae(PsaA and PavA),existed in S.marmotae sp.nov.,S.himalayana sp.nov.also.In addition,we also found other related pathogenic factors,such as autolyzed,endotoxin,cytotoxic and virulence factor regulation of protein expression.Further studies are required to define its pathogenic mechanism and potentially pathogenic to human. |
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