环境军团菌的分离培养及鉴定方法探讨

Research on the procedure for recovery and species identification of Legionella from surface environmental water

目的建立地表环境水军团菌的分离培养及种属鉴定方法。方法在广州市内8个公园中采取了44份地表湖泊水样，进行军团菌的分离培养。同一湖泊来源的分离株以扩增片段长度多态性（amplified fragment length polymorphism，AFLP）进行同源性分析；进一步的种属鉴定采用生化血清学鉴定、细胞脂肪酸成分分析、16SrRNA基因测序、巨噬细胞感染增强蛋白（mip）基因测序等多种手段相结合的方法进行分析和比较。结果8个公园内的27份水样分离出军团菌，阳性率为61．36％。初步分离的108个菌株，经AFLP同源性鉴定，得到不同菌株31株，包括嗜肺军团菌（Legionella pneumophila）20株，橡树岭军团菌（Legionella oakridgensis）1株，圣海伦军团菌（Legionella sainthelensi）1株，长滩军团菌（Legionella longbeachae）4株，菲氏军团菌（Legionella feeleii）5株。其中，嗜肺军团菌可直接通过常规的生化实验、血清学鉴定得到种属鉴定结果。但包括细胞脂肪酸成分分析在内的表型鉴定方法，均存在不同程度的不稳定性因素。结论生化实验、血清学鉴定、细胞脂肪酸成分分析等能够为军团菌的分类学鉴定提供有价值的资料。

Objective To establish a set of procedure for recovery and species identification of Legionella from the surface environmental water. Methods Forty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEα plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism （AFLP） method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing. Results Legionella was recovered among 27 （61.36%） samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of Lfeeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition. However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis. Conclusion The taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.