中国城市自然水环境中军团菌分布情况调查及嗜肺军团菌的遗传多态性研究

发布时间：2017-12-27 22:01

本文关键词：中国城市自然水环境中军团菌分布情况调查及嗜肺军团菌的遗传多态性研究　出处：《中国人民解放军军事医学科学院》2015年博士论文　论文类型：学位论文

【摘要】：军团菌是革兰氏阴性杆菌,是军团菌病的病原菌。自1979年正式命名以来,军团菌属已发现58个种,70多个血清型,20多个种可以引起人类疾病,其中嗜肺军团菌引起的病例约占临床病例的90%。军团菌广泛分布于各种水环境中,包括江、河、湖泊、池塘、温泉、地下水等自然水环境和空调冷却塔、自来水、空气加湿器、人工喷泉等人工水环境。由于军团菌病不能在人与人之间传播,只能由环境传播到人类,因此加强军团菌环境菌株的研究,对于军团菌病的溯源,研究临床菌株与环境菌株之间的关系等都有重要意义。目前对军团菌的研究主要集中于临床菌株和人工环境菌株,很少有研究关注自然水环境菌株。然而,对军团菌在其自然栖息地分布及遗传多态性的研究同样具有重要意义:首先,在我国缺少全国性的军团菌病监控系统,军团菌病的流行病资料非常匮乏,通过对自然水环境中的军团菌进行研究,可以了解自然水环境中军团菌的组成分布,对于预防和控制军团菌病的暴发流行有重要意义。其次,随着城市工业化进程的推进,自然水环境会越来越多地受到人类活动的影响,从而增加了自然水环境中军团菌作为潜在传染源的几率,了解自然水环境中军团菌的分布情况有助于军团菌病的预防和控制。第三,对自然水环境中嗜肺军团菌的种群结构及系统发育关系进行研究,有助于我们了解嗜肺军团菌的进化过程,为制定军团菌病的防治措施提供帮助。鉴于以上原因,我们在31个省、自治区、直辖市的31个城市的湖泊以及流经城市的主要江河等自然水环境中采集了水样,进行了军团菌的分离与鉴定。并对其中140株嗜肺军团菌进行了SBT分型,分析了其种群结构、系统发育关系及基因的重组,并与来自于人工环境中的菌株进行了比较。希望通过本研究,能够了解我国城市自然水环境中军团菌的组成及军团菌在各个城市中的分布,并对我国自然水环境中嗜肺军团菌的群体遗传学结构有一个初步了解,为我国城市中军团菌病的预防和控制提供有益信息。2010年7月至2014年9月,在我国31个城市采集自然水样,每个城市选取5-8个采样点,每个采样点在不同的区域采集1-5份水样,共采集水样550份。采用离心浓缩(Centrifugation enrichment,CE)、膜过滤浓缩(Filtration enrichment,FE)和热富集浓缩(Heat enrichment,HE)三种方法对水样进行浓缩处理后,进行分离培养,疑似菌株通过mip基因测序和16S r RNA测序进行鉴定。经热富集浓缩后,550份水样中有171份分离出了军团菌,而离心浓缩和膜过滤浓缩处理的水样中,分别有74份和54份培养出了军团菌。热富集浓缩处理方法的培养阳性率显著高于离心浓缩和膜过滤浓缩处理水样的培养阳性率(31.1%vs.13.1%,p0.001;31.1%vs.9.8%,p0.001,卡方检验)。三种水样处理方法对四种军团菌(嗜肺军团菌、长滩军团菌、菲氏军团菌和吉斯特军团菌)的分离率产生了影响,且差异显著(Fisher精确检验,P0.05)。温度会对分离阳性率产生影响,经spearman相关性分析发现,分离阳性率与水样温度之间呈正相关(ρ=0.929,P=0.003)。去除冗余菌株后,共获得404株军团菌,分属于23个军团菌种,其中嗜肺军团菌146株,占36.1%,是最常见的军团菌。在非嗜肺军团菌中,长滩军团菌、菲氏军团菌和戈尔曼军团菌最常见,分别占15.3%、12.6%和12.4%。在采集水样的31个城市中,29个城市的水样分离到了军团菌,嗜肺军团菌最常见,在25个城市的自然水环境中均有发现,长滩军团菌、菲氏军团菌和戈尔曼军团菌分别在21,22和20个城市的自然水环境中存在,且军团菌的种类在各个城市间的分布存在着差异。在239份阳性水标本中,75份同时存在两种以上(含两种)的不同军团菌,其中31份水标本存在三种以上(含三种)不同的军团菌,8份标本同时分离出了4种不同的军团菌。我们的结果显示在中国的自然水环境中,军团菌普遍存在且具有高度的多态性。我们同时对其中的140株嗜肺军团菌进行了序列分型(sequence-based typing,SBT)。140株嗜肺军团菌可分为84个序列型(STs),多样性指数(the index of diversity,IOD)为0.988(95%CI:0.983-0.992),84个序列型中有31个序列型与EWGLI的SBT数据库中已有序列型相同,53个为首次发现的新序列型,同时还发现11个新的等位基因。在84个序列T型中ST1417,ST1923和ST1968分别有6株嗜肺军团菌,ST1923和ST1968仅在海南发现,而ST1417的6株嗜肺军团菌分别分离自6个不同的城市。20个序列型包含两株以上的嗜肺军团菌,另外61个序列型仅含有1株嗜肺军团菌。对140株嗜肺军团菌进行的种群结构分析发现了10个克隆复合体(clonal complexes,CCs)和47个singletons,其中CCI为中国自然水环境中的优势克隆复合体,包括分别属于10个序列型来自于9个城市的21株嗜肺军团菌,占所有菌株的15%,经过查询EWGLI的SBT数据库,CCI中10个序列型中有两个(ST239和ST1439)为致病序列型,提示CCI菌株可能会引起疾病。而中国人工环境中最常见的ST1-克隆复合体在自然水环境中并不是很常见,仅在5个城市分离的7株嗜肺军团菌属于该克隆复合体。另外,10株携带完整lag-1基因的菌株不在任何一个克隆复合体内,提示在自然水环境中,高致病力菌株并不占优势。在本研究的140株自然水环境嗜肺军团菌中,虽然未发现基因内重组事件,但发现了12个基因间重组事件:在所有的84个序列型中,74个序列型发生了基因间重组,而且其中有17个序列型发生了两次重组事件。通过比较发现,不同环境与地区菌株在分子水平上的差异并不显著,但自然水环境菌株与人工水环境菌株之间却存在着显著的差异。我们的结果显示在中国的自然水环境中,嗜肺军团菌具有高度的遗传多态性且重组频繁,不同环境及地区的自然水环境嗜肺军团菌在分子水平上差异并不显著,但与人工水环境中的菌株有显著差异。
[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
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378

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