嗜肺军团菌分子分型研究进展

军团菌病主要由嗜肺军团菌引起,尤其以血清1型为多见。在军团菌病暴发流行时,为了了解环境标本与病人标本来源的分离株的亲缘关系,需要对其进行分型。目前主要有表型分型和分子分型。表型分型只能了解最基本的分型信息,而新近发展起来的基于DNA水平的分子分型在疫情的溯源及细菌群体进化研究中发挥着越来越重要的作用。该文对嗜肺军团菌分子分型方法进行综述。     

嗜肺军团菌不同分子分型方法检测

目的采用2种分子分型方法对冷却塔水中嗜肺军团菌进行检测分析。方法分别应用扩增军团菌巨噬细胞感染力增强因子基因(Macrophage Infectivity Potentiator,MIP)后测序的分型方法以及脉冲场凝胶电泳(PFGE)对48株嗜肺军团菌进行分型。结果MIP分型方法将48株嗜肺军团菌分为5类,分别为,i>L.pnuemophile-phil-1、L.pnuemophile-phil-sg3、L.pnuemophile-phil-D-15、L.pnuemophile-phil-sg10、L.pnuemophile-phil-97-2898,其中L.pnuemophile-phil-1为优势菌群,占60.4%。PFGE分型方法将48株嗜肺军团菌分为23类,无优势菌群。结论PFGE分型方法具有更高的分型能力,更有利于流行病学溯源、调查。     

工业循环冷却塔水嗜肺军团菌分离株分子生学特性研究

目的 了解钢铁企业工业循环冷却塔水中的嗜肺军团菌污染状况及菌株分子生物学特性.方法 于2011年3月-2012年9月对邯郸某钢铁厂相对固定的车间冷却塔水进行连续监测,进行嗜肺军团菌的定量分离培养,对分离到的菌株进行血清分型、脉冲场凝胶电泳分型、基因序列分型、毒力基因(lvh、rtxA)检测.结果 共检测117份水样,其中20份水样检出嗜肺军团菌,检出率为17.1％;菌落总数为100～50000 CFU/L,中位数为12000 CFU/L;共分离到嗜肺军团菌23株,血清型包括Lp1、Lp3、Lp5、Lp6、Lp8,并以Lp1为主(占36.1％,9/23).23株嗜肺军团菌PFGE分型得到19种不同带型,其中15种带型为国内首次发现.23株嗜肺军团菌分成20个SBT型别,有12个型别为本研究新发现.23株菌中,lvh、rtxA基因阳性的分别有20、22株,两种毒力基因全部阳性的有19株.结论 钢铁企业的冷却塔水存在嗜肺军团菌污染,菌型呈现基因多态性,同时具有独特的基因组成,并普遍具有毒力.     

嗜肺军团菌基因检测及分子特征研究

目的:了解外环境水中分离的嗜肺军团菌菌株的基因特征及遗传背景。方法:40株分离株分别经血清学凝集试验、胶乳凝集试验确定为LP1型嗜肺军团菌后,利用普通PCR技术对军团菌属5S rRNA基因和嗜肺军团菌mip毒力基因的特异序列进行扩增,应用实时荧光定量PCR技术扩增嗜肺军团菌mip基因,使用脉冲场凝胶电泳(PFGE)分型技术对菌株进行全染色体DNA酶切电泳分型,并用BioNumerics Version 4.0软件进行聚类分析。结果:所有的菌株均带有属特异性5S rRNA基因及mip毒力基因,PFGE聚类分析结果显示大多数菌株的遗传相似度有一定差异。结论:所有菌株经普通PCR或定量PCR均相应扩增出其特征基因,PFGE聚类分析结果提示浙江省散在水系中分离的嗜肺军团菌菌株存在遗传多样性。     

石家庄市集中空调冷却塔水中嗜肺军团菌的基因序列分型研究

目的采用基因序列分型方法(sequence-based typing,SBT)研究石家庄市集中空调冷却塔水中军团菌基因特征。方法将石家庄市集中空调冷却塔水中分离出的35株Lp1血清型嗜肺军团菌提取基因组DNA,选取嗜肺军团菌7个管家基因flaA、pilE、asd、mip、mompS、proA、neuA进行PCR扩增,将扩增产物纯化后测序。测序结果上传欧盟军团菌感染工作组(EWGLI)数据库进行比对,得到基因型别(sequence type,ST),对结果进行基因序列分型和系统进化分析。结果 35株Lp1血清型嗜肺军团菌共分为4个ST型,其中1株由于neuA基因不能被扩增而未分型;1株为新的ST型,已获EWGLI分配序号(ST1021);其余为ST1型32株,ST345型1株。结论石家庄市集中空调冷却塔水中嗜肺军团菌分离株的流行以ST1型为主,ST345型和ST1021型为特有型别。SBT分型可以作为研究嗜肺军团菌进化关系的重要手段之一。     

石家庄市水环境中嗜肺军团菌AFLP分型研究

目的 对石家庄市水环境中分离到的嗜肺军团菌进行分子分型,研究嗜肺军团菌AFLP分型特征,为建立嗜肺军团菌分子型别库提供资料.方法 运用欧洲军团菌感染工作组(European Working Group on Legionella Infection,EWGLI)组织制定的扩增片断长度多态性分型(amplified fr...     

石家庄市集中空调系统水中嗜肺军团菌的脉冲场凝胶电泳指纹图谱分析

目的 对石家庄市集中空调系统水中分离的25株嗜肺军团菌进行脉冲场凝胶电泳(PFGE)分析,初步建立石家庄市嗜肺军团菌的PFGE分型数据库.方法 采用PFGE技术,对2008年石家庄市集中空调系统水分离的嗜肺军团菌用AscI酶切,将PFGE图像用BioNumerics 4.0软件处理后,采用统一的分子量标准对照沙门菌(B...     

嗜肺军团菌荧光定量PCR检测方法的建立

目的：建立嗜肺军团菌mip基因荧光定量PCR检测方法。方法：利用Primer Express软件设计特异性引物和探针,对质粒标准品、嗜肺军团菌、非嗜肺军团菌及其他菌株进行检测,验证方法的特异性、敏感性和重复性,最后采集环境样本进行检测。结果：该方法特异性好,嗜肺军团菌呈现阳性结果,而非嗜肺军团菌及其他菌株均为阴性结果。检测灵敏度达293copies／μl;重复性较好,Ct值变异系数较小;检测的12份环境样本中5份阳性。结论：该方法快速且具有较好的特异性、敏感性、重复性,适于外环境嗜肺军团菌污染调查及应急事件的快速检测。     

嗜肺军团菌荧光定量PCR检测

目的 建立特异、快速、敏感的TaqMan探针荧光定量PCR方法,用于检测嗜肺军团菌mip基因。方法 嗜肺军团菌及其他军团菌DNA模板来自中国疾病预防控制中心呼吸道室,嗜肺军团菌地方株及其他对照株由本中心菌种室供给。利用嗜肺军团菌mip基因保守序列设计引物和TaqMan探针,对其进行筛选,同时对荧光定量PCR反应条件和反应体系进行优化,并对嗜肺军团菌、非嗜肺军团菌及其他细菌进行检测,验证该方法的特异性、敏感性、重复性等。结果 该方法在检测多株嗜肺军团菌时,均出现阳性信号,而对非嗜肺军团菌和其他细菌则未有阳性扩增结果出现。检测灵敏度达10个拷贝/反应,从菌株核酸的提取至检测完成仅需2 h左右,可减少常规PCR操作的污染机会。结论 TaqMan荧光定量PCR方法可定量检测嗜肺军团菌,具有特异性高、快速、敏感等特点,适于外环境污染源状况的调查及应急事件的快速定量检测。     

非嗜肺军团菌的分离培养与血清分型研究

目的对吉林省某集中空调冷却塔出水口的水进行军团菌分离培养及血清学分型。方法用分离培养法检测军团菌,再用乳胶凝集对其进行血清学分型。结果在某集中空调冷却塔出水口的水中成功检测出非嗜肺军团菌(博杰曼军团菌),在吉林省属首次报道。结论利用本方法可以较好地分离培养出冷却塔出水口的水中军团菌及进行血清血分型。     

我国九省（市、区）82株嗜肺军团菌血清1型菌株的序列分型

目的 了解我国环境水中嗜肺军团菌血清1型（Lp1）菌株的序列分型特征,并初步建立我国军团菌序列分型数据库。方法 采用序列分型(SBT)方法,对我国9个省（市、自治区）2005-2008年间环境水中分离的82株Lp1菌株进行分型,同时采用脉冲场凝胶电泳(PFGE)分型方法对这些菌株进行分型,并采用BioNumerics 5.1软件对2种方法的分型结果进行聚类分析和比较。结果 82株Lp1菌株分为22种序列(ST)型,其中17种ST型是新序列型,新发现1个等位基因;ST-1型为主要的序列型,在8个省（市、自治区）均有发现,该型菌株占所有菌株的46.3％ (38/82);ST-1、ST-150、ST-154、ST-159、ST-160和ST-630出现于2个以上的分离地点,5个分离位点(B4、B5、B6、S3和S8)发现2种以上ST型;通过聚类分析,15种ST型被分为3个序列群（ST-1序列群、ST-154序列群和ST-149序列群）,其余7种ST型没有序列群归类。采用PFGE分型方法,这些菌株可被分为46种带型。SBT和PFGE两种方法结合可将82株菌株分为54种分子型别。两种方法的聚类分析结果具有良好的一致性。结论 我国环境水中Lp1菌株具有独特的SBT分布;通过研究,初步建立了我国军团菌序列分型数据库。     

Genetic diversity of Legionella pneumophila in hospital water systems

It has been shown that different patients who had acquired legionellosis in a hospital setting were infected with the same strain even years apart. However, there are no longitudinal data describing the molecular epidemiology of Legionella pneumophila strains that contaminate a water system. This raised the question if there are any shifts of L. pneumophila strains over time, or after carrying out control measures. Using genotyping on a large collection of isolates, we investigated in a retrospective study the distribution of L. pneumophila serogroups and PFGE types in six different hospitals of the University of Heidelberg between 1991 and 2001. A total of 2012 water samples were drawn for routine testing and for evaluation of control measures, 747 samples were positive for L. pneumophila. Serogroups were determined by latex agglutination or by direct fluorescence assay; and 515 L. pneumophila isolates from water systems and six from patients underwent PFGE typing after SfiI-restriction. We identified seven serogroups and 19 genotypes among the water isolates. Each hospital had one to four predominating PFGE types that were stable over the investigation period. The oldest buildings in hospitals 4 and 5 (built 1876 and 1907) had more types than the newest one (built 1986). In all hospitals PFGE types were identified that could be found only sporadically. Although each hospital had its own warm water supply, we identified types that could be found in more than one hospital. However, there was no overlap of types in buildings that were fed from different wells. Infrequently occurring nosocomial legionellosis (n=3) were only caused by predominant strains. Contamination of water supplies seemed to be dominated by stable genotypes, even after various control measures. Additional genotypes could be isolated sporadically, however, their pathogenetic relevance seemed to be questionable.     

浙江省61株嗜肺军团菌血清1型菌株的SBT序列分型

目的了解浙江省空调冷却塔/冷凝水中嗜肺军团菌血清1型(Lp1)菌株的序列分型特征。方法选取2005～2011年间浙江省10个地区空调冷却塔/冷凝水中分离的61株Lp1型嗜肺军团菌菌株,采用欧盟军团菌感染工作组(EWGLI)军团菌序列分型(SBT)方法,PCR扩增7对管家基因,对扩增产物进行测序、比对、递交,以获取等位基因号以及SBT序列型,运用DNAsp 5.0软件对测序结果进行核苷酸多态性分析,采用SplitsTree及eBURST软件对分型结果进行聚类分析。结果 61株Lp1型嗜肺军团菌可分为11种SBT序列型,其中5株(5/61)均为新发现序列型;ST-1型在10个地区(市)的冷却塔/冷凝水中均有分布,该序列型菌株占81.9%(50/61)。7个管家基因的核苷酸多态性(Pi)范围为0.01095(mip)～0.05355(pilE)。聚类分析显示,浙江省Lp1型菌株分属多个克隆系,但主要分属ST-1序列群、ST-154序列群和ST-149三大序列群,另有1株为不属于任何序列群的单体。结论聚类分析表明浙江省空调冷却塔/冷凝水中分离的Lp1型嗜肺军团菌存在遗传多样性,序列群分布与国内部分省份的结果相似,以ST-1序列型为主。     

酒店浴室花洒头上检出嗜肺军团菌的报告

目的 了解上海市奉贤区部分公共场所环境中军团菌的污染状况,为军团菌病的预防控制提供依据.方法 采集该区使用集中式空调系统的大型酒店及超市的环境样品共80份进行军团菌检测,将聚合酶链反应(PCR)技术和常规方法结合,样品先进行PCR检测,阳性样品再进行常规分离和鉴定.结果 从某酒店的浴室花洒环节标本中检出2株嗜肺军团菌.结论 环境中有嗜肺军团菌存在,易受到感染,存在隐患.     

Molecular Typing in Public Health Laboratories: From an Academic Indulgence to an Infection Control Imperative

Using three Austrian case studies, the variegated applications of molecular typing in today''s public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.