【摘要】：本課題圍繞“動物源病原體的發(fā)現(xiàn)及其對人類致病性”這一科學問題,以我國青藏高原喜馬拉雅旱獺呼吸道攜帶的病原體譜系為研究目標,將現(xiàn)場流行病學調查與實驗室檢測分析相結合,采用PacBio高通量測序技術、微生物培養(yǎng)組學技術和未知新病原鑒定技術相結合,對喜馬拉雅旱獺呼吸道菌群開展研究,分離獲得三個鏈球菌新種,分別命名為旱獺鏈球菌(Streptcoccus marmotae sp.nov.),喜馬拉雅鏈球菌(Streptcoccus himalayana sp.nov.)和耐鹽鏈球菌(Streptcoccus halotolerans sp.nov.)。通過研究初步了解了喜馬拉雅旱獺呼吸道菌群分布特征;明確了三個鏈球菌新種的準確分類地位以及與近源種的生物系統(tǒng)學關系;并對其全基因組特征、毒力相關因子開展了比較基因組學的分析。本課題對喜馬拉雅旱獺呼吸道樣本采用擴增16S rRNA序列,然后克隆轉化,blast比對,對其呼吸道菌群展開分析。結果表明:鏈球菌屬是喜馬拉雅旱獺呼吸道的主要菌群,占菌群結構的49.8%;其次為埃希菌屬和韋榮菌屬,分別占菌群結構的11.1%和8.5%;此外,在所有未知新種的克隆中,疑似鏈球菌屬新種的克隆數(shù)占69.1%,提示喜馬拉雅旱獺呼吸道菌群中分布大量未知鏈球菌新種,為進一步分離培養(yǎng)提供了理論依據(jù)。以此為線索,我們應用微生物培養(yǎng)組學方法對75份喜馬拉雅旱獺氣管黏膜標本進行篩查,成功分離到三個疑似鏈球菌新種。經(jīng)形態(tài)學觀察、生化反應比較,并結合分子生物學鑒定,該三個未知新種均歸屬為鏈球菌屬,根據(jù)三個未知新種的特征,依照國際微生物命名標準,分別將其命名為旱獺鏈球菌(S.marmotae sp.nov.),喜馬拉雅鏈球菌(S.himalayana sp.nov.)和耐鹽鏈球菌(S.halotolerans sp.nov.)。三個鏈球菌新種在血平板上37℃培養(yǎng)24 h后形成直徑0.5-1.0 mm,灰白色、圓形隆起、濕潤、邊緣整齊的針尖狀菌落,a溶血。單個菌體呈圓形或橢圓形,數(shù)個菌體排列呈鏈狀,革蘭染色陽性,無芽胞,無鞭毛。三個鏈球菌新種均在22、30、37、42°C培養(yǎng)時生長,4°C、15°C時不生長。其中喜馬拉雅鏈球菌和旱獺鏈球菌在含2.5%NaCl的腦心羊血培養(yǎng)基上生長,在含4.5%、6.5%NaCl的腦心羊血培養(yǎng)基上不生長。但是耐鹽鏈球菌在含9.0%NaCl的腦心羊血培養(yǎng)基上仍可以生長。三個鏈球菌新種的生化反應結果明顯區(qū)別于同屬內(nèi)其他近源種�；�16S rRNA序列、3個保守基因(sodA、rpoB和groEL)和全基因組的系統(tǒng)發(fā)生關系分析表明,旱獺鏈球菌與S.suis,S.ovis和S.minor親緣關系較近,歸屬為鏈球菌屬豬鏈群;喜馬拉雅鏈球菌與S.acidominimus,S.cuniculi和S.marmotae有較近的進化關系,也屬于鏈球菌屬豬鏈群;耐鹽鏈球菌與變異群的S.thoraltensis,S.hyovaginalis聚在一起,歸屬為鏈球菌屬變異群。通過與GenBank中其他鏈球菌已知種進行DNA-DNA在線雜交,發(fā)現(xiàn)三個鏈球菌新種與其他鏈球菌已知種相似性均低于70%。旱獺鏈球菌基因組全長2,322,791 bp,G+C含量為41.6 mol%,編碼2,377個基因,攜帶58個tRNAs,12個rRNAs,2個質粒(大小分別為3,762 bp和5,997bp),11個基因組島。喜馬拉雅鏈球菌基因組全長2,275,471 bp,G+C含量為41.3mol%,編碼2,195個基因,攜帶57個tRNAs,12個rRNAs,6個基因組島。耐鹽鏈球菌基因組全長1,823,556 bp,G+C含量為39.9mol%,編碼2,068個基因,攜帶68個tRNAs,18個rRNAs,9個基因組島。通過將三種鏈球菌新種的基因組序列與VFDB數(shù)據(jù)庫收錄的毒力基因進行BLAST比對分析,結果表明Has(Hyaluronic acid capsule),ClpC(Caseinolytic protease C)和ClpE(Caseinolytic protease E)是三個鏈球菌新種共有的毒力因子;作為肺炎鏈球菌主要侵襲力的表面黏附素PsaA(pneumococcal surface adhesin A)和PavA(pneumococcal adherence and virulence factor A),在旱獺鏈球菌和喜馬拉雅鏈球菌中都存在;除此以外,VFDB數(shù)據(jù)庫中編碼細胞毒素、自溶素的基因在三個鏈球菌新種基因組中也存在,三個鏈球菌新種是否對動物和人具有致病性,其致病機制如何,是我們接下來要進行的課題研究。
[Abstract]:Focusing on the scientific problem of "discovery of animal-derived pathogens and their pathogenicity to humans", this paper takes the respiratory tract pathogen pedigree of Himalayan marmots from the Qinghai-Tibet Plateau as the research object, combines field epidemiological investigation with laboratory detection and analysis, and adopts PacBio high-throughput sequencing technology to study the microbial culture group. Three new strains of Streptococcus were isolated from the respiratory tract of Marmota himalayana sp. nov., Streptcoccus himalayana sp. Nov. and Streptcoccus halotolerans sp. Nov. respectively. The distribution characteristics of respiratory tract microflora of Himalayan marmots have been preliminarily understood, the accurate taxonomic status of three new species of Streptococcus and the relationship between the three new species and their biological systems have been clarified, and the comparative genomics analysis of the whole genome characteristics and virulence-related factors has been carried out. 16S rRNA sequence was amplified, cloned and transformed, and blast was used to analyze the respiratory tract microflora. The results showed that Streptococcus was the main microflora in the respiratory tract of Himalayan marmots, accounting for 49.8% of the microflora structure, followed by Escherichia coli and Virginia, accounting for 11.1% and 8.5% of the microflora structure, respectively. In addition, among all the clones of unknown new species, it was suspected. The clones of new species of Streptococcus accounted for 69.1%, suggesting that a large number of new species of unknown Streptococcus were distributed in the respiratory tract flora of Himalayan marmots, providing a theoretical basis for further isolation and culture. Based on this clue, 75 tracheal mucosa specimens of Himalayan marmots were screened by microbiology and three were successfully isolated. A new species of suspected Streptococcus was identified as S. marmotae sp. Nov. and S. himalayana sp. according to the characteristics of the three unknown new species and according to the international microbiological nomenclature standards, they were named S. marmotae sp. Nov. and S. himalayana sp. respectively. S. halotolerans sp. Nov. and S. halotolerans sp. Nov. Three new species of Streptococcus were cultured on the blood plate at 37 C for 24 hours and then formed 0.5-1.0 mm in diameter, gray-white, round protuberance, moist, well-margined, needle-shaped colony, a hemolysis. A single bacteria was round or oval, several bacteria arranged in a chain shape, Gram-positive, sporeless, flagella-free. Streptococcus himalayanus and Streptococcus Marmota grew on the medium containing 2.5% NaCl and did not grow on the medium containing 4.5% and 6.5% NaCl. But Streptococcus halotolerans could still grow on the medium containing 9.0% NaCl. The results of biochemical reactions of three new Streptococcus species were significantly different from other near source species. Based on 16S rRNA sequences, 3 conserved genes (sodA, rpoB and groEL) and genome wide phylogenetic relationships showed that Streptococcus marmos was closely related to S.suis, S.ovis and S.minor and belonged to Streptococcus group *; Himalaya. Streptococcus has a closer evolutionary relationship with S.acidominimus, S.cuniculi and S.marmotae, and also belongs to Streptococcus * group. Streptococcus salt resistant Streptococcus is clustered together with S.thoraltensis and S.hyovaginalis of the mutant group and belongs to the group of Streptococcus mutans. By means of DNA-DNA online hybridization with other known species of Streptococcus in GenBank, three Streptococcus were found. The genome of Streptococcus Marmota was 2,322,791 BP in length, with a G+C content of 41.6 mol%, encoding 2,377 genes, carrying 58 tRNAs, 12 rRNAs, 2 plasmids (3,762 BP and 5,997 BP respectively) and 11 genomic islands. The genome of Streptococcus himalayae was 2,275,471 BP in length and 41.3 m in G+C content. Ol%, encoding 2,195 genes, carrying 57 tRNAs, 12 rRNAs, 6 genomic islands. The genome of S. halotolerans was 1,823,556 BP in length, containing 39.9 mol% of G + C, encoding 2,068 genes, carrying 68 tRNAs, 18 rRNAs, and 9 genomic islands. BLAST ratios were made between the genomic sequences of the three new species of S. halotolerans and the virulence genes in the VFDB database. The results showed that Has (Hyaluronic acid capsule), ClpC (Caseinolytic protease C) and CPE (Caseinolytic protease E) were the common virulence factors of three new species of Streptococcus, and the surface adhesins PsaA (pneumococcal surface adhesin A) and PAVA (pneumococcal adherence and virulence factor) were the main invasive factors of Streptococcus pneumoniae. In addition, cytotoxins are encoded in the VFDB database, and autolysin genes are also present in the genomes of three new species of Streptococcus. Whether the three new species of Streptococcus are pathogenic to animals and humans, and how their pathogenic mechanisms are, are the topics we will study next.
【學位授予單位】：山西大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.611

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