本文關(guān)鍵詞：中國城市自然水環(huán)境中軍團(tuán)菌分布情況調(diào)查及嗜肺軍團(tuán)菌的遺傳多態(tài)性研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 軍團(tuán)菌 嗜肺軍團(tuán)菌 序列分型 群體遺傳學(xué)

【摘要】：軍團(tuán)菌是革蘭氏陰性桿菌,是軍團(tuán)菌病的病原菌。自1979年正式命名以來,軍團(tuán)菌屬已發(fā)現(xiàn)58個(gè)種,70多個(gè)血清型,20多個(gè)種可以引起人類疾病,其中嗜肺軍團(tuán)菌引起的病例約占臨床病例的90%。軍團(tuán)菌廣泛分布于各種水環(huán)境中,包括江、河、湖泊、池塘、溫泉、地下水等自然水環(huán)境和空調(diào)冷卻塔、自來水、空氣加濕器、人工噴泉等人工水環(huán)境。由于軍團(tuán)菌病不能在人與人之間傳播,只能由環(huán)境傳播到人類,因此加強(qiáng)軍團(tuán)菌環(huán)境菌株的研究,對于軍團(tuán)菌病的溯源,研究臨床菌株與環(huán)境菌株之間的關(guān)系等都有重要意義。目前對軍團(tuán)菌的研究主要集中于臨床菌株和人工環(huán)境菌株,很少有研究關(guān)注自然水環(huán)境菌株。然而,對軍團(tuán)菌在其自然棲息地分布及遺傳多態(tài)性的研究同樣具有重要意義:首先,在我國缺少全國性的軍團(tuán)菌病監(jiān)控系統(tǒng),軍團(tuán)菌病的流行病資料非常匱乏,通過對自然水環(huán)境中的軍團(tuán)菌進(jìn)行研究,可以了解自然水環(huán)境中軍團(tuán)菌的組成分布,對于預(yù)防和控制軍團(tuán)菌病的暴發(fā)流行有重要意義。其次,隨著城市工業(yè)化進(jìn)程的推進(jìn),自然水環(huán)境會(huì)越來越多地受到人類活動(dòng)的影響,從而增加了自然水環(huán)境中軍團(tuán)菌作為潛在傳染源的幾率,了解自然水環(huán)境中軍團(tuán)菌的分布情況有助于軍團(tuán)菌病的預(yù)防和控制。第三,對自然水環(huán)境中嗜肺軍團(tuán)菌的種群結(jié)構(gòu)及系統(tǒng)發(fā)育關(guān)系進(jìn)行研究,有助于我們了解嗜肺軍團(tuán)菌的進(jìn)化過程,為制定軍團(tuán)菌病的防治措施提供幫助。鑒于以上原因,我們在31個(gè)省、自治區(qū)、直轄市的31個(gè)城市的湖泊以及流經(jīng)城市的主要江河等自然水環(huán)境中采集了水樣,進(jìn)行了軍團(tuán)菌的分離與鑒定。并對其中140株嗜肺軍團(tuán)菌進(jìn)行了SBT分型,分析了其種群結(jié)構(gòu)、系統(tǒng)發(fā)育關(guān)系及基因的重組,并與來自于人工環(huán)境中的菌株進(jìn)行了比較。希望通過本研究,能夠了解我國城市自然水環(huán)境中軍團(tuán)菌的組成及軍團(tuán)菌在各個(gè)城市中的分布,并對我國自然水環(huán)境中嗜肺軍團(tuán)菌的群體遺傳學(xué)結(jié)構(gòu)有一個(gè)初步了解,為我國城市中軍團(tuán)菌病的預(yù)防和控制提供有益信息。2010年7月至2014年9月,在我國31個(gè)城市采集自然水樣,每個(gè)城市選取5-8個(gè)采樣點(diǎn),每個(gè)采樣點(diǎn)在不同的區(qū)域采集1-5份水樣,共采集水樣550份。采用離心濃縮(Centrifugation enrichment,CE)、膜過濾濃縮(Filtration enrichment,FE)和熱富集濃縮(Heat enrichment,HE)三種方法對水樣進(jìn)行濃縮處理后,進(jìn)行分離培養(yǎng),疑似菌株通過mip基因測序和16S r RNA測序進(jìn)行鑒定。經(jīng)熱富集濃縮后,550份水樣中有171份分離出了軍團(tuán)菌,而離心濃縮和膜過濾濃縮處理的水樣中,分別有74份和54份培養(yǎng)出了軍團(tuán)菌。熱富集濃縮處理方法的培養(yǎng)陽性率顯著高于離心濃縮和膜過濾濃縮處理水樣的培養(yǎng)陽性率(31.1%vs.13.1%,p0.001;31.1%vs.9.8%,p0.001,卡方檢驗(yàn))。三種水樣處理方法對四種軍團(tuán)菌(嗜肺軍團(tuán)菌、長灘軍團(tuán)菌、菲氏軍團(tuán)菌和吉斯特軍團(tuán)菌)的分離率產(chǎn)生了影響,且差異顯著(Fisher精確檢驗(yàn),P0.05)。溫度會(huì)對分離陽性率產(chǎn)生影響,經(jīng)spearman相關(guān)性分析發(fā)現(xiàn),分離陽性率與水樣溫度之間呈正相關(guān)(ρ=0.929,P=0.003)。去除冗余菌株后,共獲得404株軍團(tuán)菌,分屬于23個(gè)軍團(tuán)菌種,其中嗜肺軍團(tuán)菌146株,占36.1%,是最常見的軍團(tuán)菌。在非嗜肺軍團(tuán)菌中,長灘軍團(tuán)菌、菲氏軍團(tuán)菌和戈?duì)柭妶F(tuán)菌最常見,分別占15.3%、12.6%和12.4%。在采集水樣的31個(gè)城市中,29個(gè)城市的水樣分離到了軍團(tuán)菌,嗜肺軍團(tuán)菌最常見,在25個(gè)城市的自然水環(huán)境中均有發(fā)現(xiàn),長灘軍團(tuán)菌、菲氏軍團(tuán)菌和戈?duì)柭妶F(tuán)菌分別在21,22和20個(gè)城市的自然水環(huán)境中存在,且軍團(tuán)菌的種類在各個(gè)城市間的分布存在著差異。在239份陽性水標(biāo)本中,75份同時(shí)存在兩種以上(含兩種)的不同軍團(tuán)菌,其中31份水標(biāo)本存在三種以上(含三種)不同的軍團(tuán)菌,8份標(biāo)本同時(shí)分離出了4種不同的軍團(tuán)菌。我們的結(jié)果顯示在中國的自然水環(huán)境中,軍團(tuán)菌普遍存在且具有高度的多態(tài)性。我們同時(shí)對其中的140株嗜肺軍團(tuán)菌進(jìn)行了序列分型(sequence-based typing,SBT)。140株嗜肺軍團(tuán)菌可分為84個(gè)序列型(STs),多樣性指數(shù)(the index of diversity,IOD)為0.988(95%CI:0.983-0.992),84個(gè)序列型中有31個(gè)序列型與EWGLI的SBT數(shù)據(jù)庫中已有序列型相同,53個(gè)為首次發(fā)現(xiàn)的新序列型,同時(shí)還發(fā)現(xiàn)11個(gè)新的等位基因。在84個(gè)序列T型中ST1417,ST1923和ST1968分別有6株嗜肺軍團(tuán)菌,ST1923和ST1968僅在海南發(fā)現(xiàn),而ST1417的6株嗜肺軍團(tuán)菌分別分離自6個(gè)不同的城市。20個(gè)序列型包含兩株以上的嗜肺軍團(tuán)菌,另外61個(gè)序列型僅含有1株嗜肺軍團(tuán)菌。對140株嗜肺軍團(tuán)菌進(jìn)行的種群結(jié)構(gòu)分析發(fā)現(xiàn)了10個(gè)克隆復(fù)合體(clonal complexes,CCs)和47個(gè)singletons,其中CCI為中國自然水環(huán)境中的優(yōu)勢克隆復(fù)合體,包括分別屬于10個(gè)序列型來自于9個(gè)城市的21株嗜肺軍團(tuán)菌,占所有菌株的15%,經(jīng)過查詢EWGLI的SBT數(shù)據(jù)庫,CCI中10個(gè)序列型中有兩個(gè)(ST239和ST1439)為致病序列型,提示CCI菌株可能會(huì)引起疾病。而中國人工環(huán)境中最常見的ST1-克隆復(fù)合體在自然水環(huán)境中并不是很常見,僅在5個(gè)城市分離的7株嗜肺軍團(tuán)菌屬于該克隆復(fù)合體。另外,10株攜帶完整lag-1基因的菌株不在任何一個(gè)克隆復(fù)合體內(nèi),提示在自然水環(huán)境中,高致病力菌株并不占優(yōu)勢。在本研究的140株自然水環(huán)境嗜肺軍團(tuán)菌中,雖然未發(fā)現(xiàn)基因內(nèi)重組事件,但發(fā)現(xiàn)了12個(gè)基因間重組事件:在所有的84個(gè)序列型中,74個(gè)序列型發(fā)生了基因間重組,而且其中有17個(gè)序列型發(fā)生了兩次重組事件。通過比較發(fā)現(xiàn),不同環(huán)境與地區(qū)菌株在分子水平上的差異并不顯著,但自然水環(huán)境菌株與人工水環(huán)境菌株之間卻存在著顯著的差異。我們的結(jié)果顯示在中國的自然水環(huán)境中,嗜肺軍團(tuán)菌具有高度的遺傳多態(tài)性且重組頻繁,不同環(huán)境及地區(qū)的自然水環(huán)境嗜肺軍團(tuán)菌在分子水平上差異并不顯著,但與人工水環(huán)境中的菌株有顯著差異。
[Abstract]:Legionella is gram-negative bacilli and is the pathogen of legionellosis. Since its formal designation in 1979, Legionella has been found in 58 species, more than 70 serotypes and more than 20 species, which can cause human diseases, of which 90% cases are caused by Legionella pneumophila. Legionella is widely distributed in all kinds of water environment, including natural water environment such as rivers, lakes, ponds, hot springs, underground water and air conditioning cooling towers, tap water, air humidifiers, artificial fountain and other artificial water environment. Legionellosis cannot be transmitted from person to person. It can only be transmitted to humans through environment. Therefore, strengthening the research of Legionella environmental strains is very important for tracing the origin of legionellosis and studying the relationship between clinical strains and environmental strains. At present, the study of Legionella mainly focuses on clinical strains and artificial environmental strains, and few studies have paid attention to the strain of natural water environment. However, the Legionella also has an important significance in the research of genetic polymorphism in its natural habitat and distribution of Legionella in: first, the lack of national monitoring system in our country, is the lack of epidemiological data of Legionnaires disease, through the study of the nature of Legionella in water environment, can understand the composition and distribution of Legionella in nature the water environment, has important significance for the prevention and control of epidemic outbreak of Legionnaires disease. Secondly, with the city to promote the industrialization process, the natural water environment will be more and more affected by human activities, thus increasing the Legionella natural environment as the probability of potential source of infection, investigate the distribution of Legionella in natural aquatic environment are helpful to the prevention and control of Legionella disease. Third, research on the relationship between Legionella pneumophila population structure and phylogenetic relationship in natural water environment will help us understand the evolution process of Legionella pneumophila, and provide help for formulating prevention and treatment measures of legionellosis. In view of the above reasons, we collected water samples from 31 provinces, autonomous regions and municipalities in 31 cities and lakes and other natural rivers that flow through the city, and carried out Legionella isolation and identification. 140 strains of Legionella pneumophila were genotyped by SBT. Their population structure, phylogenetic relationship and gene recombination were analyzed, and compared with those from artificial environment. Hope that through this study, to understand the composition and distribution of Legionella Legionella in China city natural water environment in each city, and have a preliminary understanding of the structure of population genetics of our natural environment of Legionella pneumophila, for our country city LP bacteria disease prevention and control useful information. From July 2010 to September 2014, natural water samples were collected from 31 cities in China. 5-8 samples were selected from each city. 1-5 samples were collected from different locations in different sampling points, and 550 samples were collected. By centrifugation (Centrifugation, enrichment, CE (Filtration) membrane for concentration of enrichment, FE (Heat) and thermal enrichment of enrichment, HE) three methods were concentrated in water samples, were cultured, suspected strains were identified by MIP 16S gene sequencing and R RNA sequencing. After thermal enrichment, 171 Legionella strains were isolated from 550 samples, while 74 and 54 of them were Legionella. The positive rate of heat enrichment treatment was significantly higher than that of centrifugation and membrane filtration treatment (31.1%vs.13.1%, p0.001, 31.1%vs.9.8%, p0.001, chi square test). Three samples of four kinds of treatment methods (Legionella pneumophila and Long Beach, Legionella pneumophila and guist Filippi Legionella) the separation rate of the impact, and the difference was significant (Fisher exact test, P0.05). The temperature will affect the positive rate of Spearman, the correlation analysis found that the separation between the positive rate and water temperature were positively correlated (=0.929, P=0.003). After the redundant strains were removed, 404 Legionella strains were obtained, which belong to 23 Legionella strains, of which 146 strains of Legionella pneumophila, accounting for 36.1%, were the most common Legionella. In the non Legionella pneumophila in Long Beach, the most common Legionella, Filippi Legionella and Goleman Legionella, accounted for 15.3%, 12.6% and 12.4%. In 31 water samples were collected in the city, 29 city water separation to Legionella pneumophila, were the most common, is found in the natural water environment in Long Beach city 25, Legionella, Filippi Legionella and Goleman respectively in the presence of Legionella 21,22 and 20 of the city's natural water environment in different types of Legionella and distributed in each city between. In 239 positive water samples, 75 samples at the same time, there are more than two kinds (including two) of Legionella, of which 31 water samples had more than three (including three) different Legionella, 8 specimens isolated from 4 different legionella. Our results show that Legionella is common and highly polymorphic in China's natural water environment. At the same time, 140 strains of Legionella pneumophila were sequenced (sequence-based typing, SBT). 140 strains of Legionella pneumophila can be divided into 84 sequence types (STs), diversity index (the index of diversity, IOD) 0.988 (95%CI:0.983-0.992), 84 sequence type in the sequence has 31 sequences and EWGLI SBT database in the same type, 53 new sequence type was first discovered at the same time, also found 11 new alleles. In the 84 sequence T, ST1417, ST1923 and ST1968 had 6 strains of Legionella pneumophila, ST1923 and ST1968 were found only in Hainan, while ST1417 6 strains of Legionella pneumophila were isolated from 6 different cities. The 20 sequence contained more than two strains of Legionella pneumophila, and the other 61 sequences contained only 1 Legionella pneumophila. The population structure analysis of 140 strains of Legionella pneumophila found 10 clone complexes (CL
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R378

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