我国部分水系和土壤中嗜肺军团菌和长滩军团菌遗传多态性研究

Legionella pneumophila and Legionella longbeachae are two of the most important pathogenic Legionella species. They can be both distributed in natural water samples and soil. Legionella pneumophila causes infections by inhalation of aerosols from contaminated water; while Legionella longbeachae can cause infections by inhalation of dust from compost, potted soil and agricultural soil. Therefore, the isolates of these two species from soil or water sources may have different virulence, and this inconsistency may be related to their environmental adaptations. This project intends to collect environmental samples from water systems of 10 important cities in China and its surrounding agricultural soil for isolation and culture of Legionella. The isolates will be identified by phenotypic analysis and rapid identification and the composition of the two species in the two environments will be determined. The isolates will be also determined by sequence-based typing method and whole genome sequencing method. The population genetic structure and phylogeny of isolates from the different environmental sources and the mechanism of the adaptation of Legionella pneumophila and Legionella longbeachae to different environments will be researched. The effects of two different environments on the distribution of the strain will be identified. This project will be helpful to understand the environmental composition, distribution and genetic polymorphism of two important pathogenic Legionella species in the water system and its surrounding agricultural soil of our country, contribute to providing scientific basis for preventing and controlling the outbreak of Legionaires' disease and explaining the mechanism of different environmental adaptability and pathogenicity of the two species.

嗜肺军团菌和长滩军团菌是两种最重要的致病性军团菌种，严重威胁民众健康。长滩军团菌与嗜肺军团菌均可分布于自然水样和土壤，嗜肺军团菌经由吸入含菌气溶胶而致病；长滩军团菌经由吸入来源于堆肥盆栽及农业土壤含菌微尘而致病，因此土壤来源或水环境来源的菌株可能存在毒力差异，且该差异与环境适应性有关。本课题拟采集我国10个重要城市水系及其周边土壤进行军团菌分离培养，通过表型分析和快速鉴定分离菌株，确定嗜肺军团菌和长滩军团菌在不同环境中的构成。采用多基因位点序列分型方法和全基因组测序方法对分离的嗜肺军团菌和长滩军团菌株进行测定，研究不同环境来源菌株的群体遗传学结构与系统发育关系；以及嗜肺军团菌和长滩军团菌环境适应性的机理。本课题的开展将有助于了解我国重要城市水系及其周边土壤中两种重要的致病性军团菌环境组成、分布和遗传多态性, 为预防和控制军团菌病暴发流行、阐述两种细菌不同环境适应性及致病性机理提供科学依据。

军团菌是一种环境来源的细菌。嗜肺军团菌（LP）是最重要的致病性军团菌种，严重威胁民众健康。LP可分布于自然水样和土壤，经由人工设备播散含菌气溶胶而致病；但是军团菌在中国自然环境中的分布及遗传多态性目前仍然没有一个完整而详细流行病学调查与研究。本研究通过对来自我国11个省份16个城市水系（共575份样品）和土壤（共442份样品）分离军团菌的研究表明 ：1）22.43%的水样品和9.28%的土壤样品中可分离到军团菌， 2）致病性军团菌种在我国水环境和土壤环境中均广泛分布；但分布不均一，且土壤具有更高的军团菌种多样性； 3）LP是我国土壤和水系中主要的致病性军团菌，在两种环境中均占所有军团菌分离株均为75%左右；4）LP sg1占我国环境中所分离LP菌株22.87%，且在水系中分布频率更高（26.66% vs.12.21%）。此外，在中国环境来源LP中还发现了许多新的序列类型（STs）。基因内重组不仅作用于水样来源LP也作用于土壤LP。系统发育、种群结构和分子进化分析表明，尽管土壤和水样分离株的遗传差异比例较小，不同环境来源LP分离株可能具有特殊的遗传背景，以更能适应土壤或水环境。我们的研究表明，土壤是中国致病性军团菌储存和传播的另一来源。未来研究应评估土壤中军团菌的存在对公共卫生的影响，并说明军团菌在水和土壤之间的遗传和致病性差异，特别是最广泛分布的LP。此外，通过分子进化、遗传多态性研究及LP感染细胞模型研究表明，土壤菌株和水样菌株的不仅具有遗传多态性，而且也具有毒力具有异质性，总体而言土壤来源菌株感染巨噬细胞后具有迅速诱导炎症因子高表达的能力，但是相对长期感染过程中，水样来源菌株可能具有更强的毒力。我们推测在感染早期土壤来源LP菌株的LPS，鞭毛蛋白，CpG DNA等通过模式识别受体激活宿主的NF-κB信号通路诱导炎症因子转录的能力可能更强。但是在感染后期，水样LP在复制小体中复制，通过T4SS分泌一系列效应蛋白可能具有更强的激活THP-1细胞NF-κB信号通路诱导炎症因子转录的能力，其机制有待进一步研究。