公共场所冷却塔水嗜肺军团菌的基因序列分型研究

目的 为了解温州市中央空调冷却塔水中嗜肺军团菌(Legionella pneumophila,LP)基因特征,采用序列分型方法(sequenced-based typing,SBT)对公共场所中央空调冷却塔水中的嗜肺军团菌进行分子分型研究。方法 选择嗜肺军团菌的7 个管家基因 flaA、asd、mip、pilE、mompS、proA 和 neuA 作为目的基因, 对温州市公共场所中央空调冷却塔水中分离的31株嗜肺军团菌进行PCR扩增并测序。测序结果上传欧盟军团菌感染工作组(EWGLI)数据库进行比对、分型。运用DNAsp 5.0 、Bionumerics5.1及SplitsTree 等软件对分型结果进行分析。结果 31株嗜肺军团菌中,11株LP1型被分为2种ST型;17株非LP1型被分为8种ST型,其中7种ST型是新发现的序列型,并新发现1个等位基因;另3株非LP1型因缺少neuA基因未被数据库确认 。7个管家基因的核苷酸多态性 (Pi)范围为0.00995(mip)～0.02311(flaA)。 flaA具有最大的核苷酸多态性 ( 0.02311 )。经聚类分析得到本地分离的嗜肺军团菌株的遗传进化关系。结论 温州市公共场所冷却塔水中的嗜肺军团菌与国内外其他地区报道的嗜肺军团菌具有一定的亲缘关系,同时也具有地区特异性和遗传多样性。

Sequence-based typing of Legionella pneumophila isolates from the water of public cooling tower

The aim was to investigate the genetic characteristics of Legionella pneumophila (LP) isolated from cooling tower water of the central air conditioning systems in public place of Wenzhou City by the sequence-based typing (SBT). Seven housekeeping genes including flaA, asd, mip, pilE, mompS, proA, and neuA were amplified by PCR from 31 strains of LP, and then the amplified products were sequenced and the results were compared with the database of EWGLI. Furthermore, genotyping results were analyzed by DNAsp 5.0, BioNumerics 5.1, and SplitsTree. Results showed that among 31 LP strains, 11 strains of LP1 were divided into 2 sequence types (STs), and 17 strains of non-LP1 were divided into 8 STs, including 7 new STs and one new allele gene. The neuA gene was not amplified from 3 strains, and hence they were not assigned to a specific genotype. The range of 7 housekeeping genes’ nucleotide polymorphism (Pi) was 0.00995 (mip) to 0.02311 (flaA) with that flaA has the highest nucleotide polymorphism. The genetic evolutionary relationship of the local isolates was obtained by using cluster analysis. In brief, L. pneumophila strains from the cooling tower water in public place in Wenzhou City are related to other domestic and foreign strains, and also have region-specific and genetic diversity.