广东省2003-2011年35株C群脑膜炎奈瑟菌多位点序列分析

流行性脑脊髓膜炎(流脑)近年来在广东省发病维持在低水平,但流行菌群有不断变迁趋势,B、C群菌株已成为主要致病菌群之一.本研究采用多位点序列分析(MLST),对2003-2011年广东省保存的35株C群脑膜炎奈瑟菌(Nm)分离株开展分子流行病学研究,探讨菌株间遗传进化关系.     

Multilocus sequence typing approach for a broader range of species of Leishmania genus: Describing parasite diversity in Argentina

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina.     

125株耐碳青霉烯类抗菌药物鲍氏不动杆菌多位点序列分型

目的了解医院耐碳青霉烯类鲍氏不动杆菌的遗传特征。方法选取耐碳青霉烯类鲍氏不动杆菌分离株,进行多位点序列分型(MLST)。结果选取的125株耐碳青霉烯类鲍氏不动杆菌,7个看家基因中6个等位基因数为1,gpi的等位基因数为3,可变位点数为3;125株菌MLST分析,3个ST型,其中ST 75型有82株,ST92型有20株,ST138型有23株,ST75、ST92与ST138属于同一克隆群。结论医院耐碳青霉烯类鲍氏不动杆菌相对进化较慢,对多种抗菌药物耐药,应严格消毒措施及无菌操作,减少由耐碳青霉烯类鲍氏不动杆菌引起的医院感染;减少抗菌药物的使用,减缓泛耐药菌的产生。     

Geographical segregation of Cryptosporidium parvum multilocus genotypes in Europe

Cryptosporidium parvum is a common enteric protozoan pathogen of humans and livestock. Multilocus genotyping based on simple sequence repeat polymorphisms has been used extensively to identify transmission cycles and to investigate the structure of C. parvum populations and of the related pathogen Cryptosporidium hominis. Using such methods, the zoonotic transmission of C. parvum has been shown to be epidemiologically important. Because different genetic markers have been used in different surveys, the comparison of Cryptosporidium genotypes across different laboratories is often not feasible. Therefore, few comparisons of Cryptosporidium populations across wide geographical areas have been published and our understanding of the epidemiology of cryptosporidiosis is fragmented. Here we report on the genotypic analysis of a large collection of 692 C. parvum isolates originating primarily from cattle and other ruminants from Italy, Ireland and Scotland. Because the same genotypic markers were used in these surveys, it was possible to merge the data. We found significant geographical segregation and a correlation between genetic and geographic distance, consistent with a model of isolation by distance. The presence of strong LD and positive I_A^S values in the combined MLG dataset suggest departure from panmixia, with different population structures of the parasite prevailing in each country.     

海南省脑膜炎奈瑟菌多位点序列分型研究

目的 了解海南省脑膜炎奈瑟菌（Nm）的分子分型特征。方法 采用多位点序列分析（MLST）技术,对2005-2015年海南省流行性脑脊髓膜炎（ECM）病人、密切接触者和健康带菌者样本中分离到的16株Nm和1份脑脊液样本进行核酸序列分析。结果 17份样本共分为11种不同的Nm ST型别,分属于ST-4821、ST-11、ST-5、ST-175、ST-364克隆群和5个无序列群分类（UA）,其中前3个克隆群为高致病性克隆群,分别占29.41%（5株）、17.65%（3株）、5.88%（1株）。ST-4821克隆群有ST-4821型和ST-3200型。ST-4821型来自3例C群ECM病例和1株不可分群密切接触者。ST-3200型来自2005年的C群健康带菌者。ST-11、ST-5克隆群分别只有W135群的ST-11型和不可分群的ST-7型,均来自健康带菌者。B群5株有5种ST,其中ST-11127型和ST-11134型为新发现的ST,2015年海口市首例B群病人是ST-7962型,与2010年分离自儋州市ECM密切接触者的B群ST-11127型同源,均为UA,其他不同基因群序列型相互间进化关系较远。结论 海南省Nm分子型别具有基因多样性,Nm C群ST-4821型是优势序列群。健康人群中携带有高致病性克隆群菌株。     

新生儿科分离白假丝酵母菌多位点序列分型研究

目的分析白假丝酵母菌多位点序列分型(MLST)与标本来源、药物敏感性之间的关系,探讨菌株间的亲缘关系及其分子流行病学特征。方法收集2015年4—5月某院新生儿科病房分离的白假丝酵母菌,采用试剂盒对菌株进行体外药物敏感性测试,并采用MLST方法对菌株进行分型,分析菌株间的同源性及分子流行病学特征。结果共分离15株白假丝酵母菌,来自4例患儿和1名医务人员,15株白假丝酵母菌经MLST获得3个序列型(ST),其中13株为ST1997,另外2株分别为ST1359和ST1933。经eBURST软件对ST分型进行聚类分析,ST1997菌株属于Group 13,ST1359菌株属于Group 1,ST1933菌株属于Group 20。同一患者外周血和外周静脉导管分离的白假丝酵母菌属于同一克隆株。结论新生儿科病房存在白假丝酵母菌血流感染的暴发,应积极制定相应的预防控制措施,加强对新生儿科病房的医院感染管理。     

食品及环境阪崎肠杆菌分离株的MLST和PFGE分型方法比较

目的:对PFGE和MLST方法在阪崎肠杆菌分型研究中的分辨力和潜在价值进行了比较研究和论述。方法:采用PFGE和MLST方法对本实验室分离的19株阪崎肠杆菌和一株标准菌株ATCC51329进行分型研究。结果:PFGE方法可将20株阪崎肠杆菌分为6大类群,共16个PFGE型,分辨力为0.9474。而MLST方法可将20株菌株分为12个ST型,分辨力为0.9263。结论:PFGE法较MLST法具有较高的分辨力,可用于阪崎肠杆菌的溯源研究,而MLST分型方法能通过全球比对数据库得到更多的关于进化和亲缘关系的分析资料,在致病研究、流行病学调查、进化研究方面优于PFGE法。     

多位点序列分型技术在深圳副溶血弧菌同源性分析中的应用研究

目的 了解副溶血弧菌临床分离株的毒力基因、血清型及基因分型特征,探讨多位点序列分型(MLST)分型技术对其同源性分析的应用价值。方法 收集深圳地区2007-2014年食物中毒及医院散发病例来源的68株副溶血弧菌菌株,对其进行血清型、毒力基因分析。选择副溶血弧菌MLST数据库提供的7个管家基因,PCR扩增测序后采用BioEdit、eBURST等软件,对本次研究的68株副溶血弧菌进行MLST同源性分析。结果 68株副溶血弧菌共得到7种不同的血清型,优势血清型为O3∶K6(47/68),其余血清型分别为O4∶K8、O5∶K68、O4∶K13等。副溶血弧菌临床分离株94%为tdh+trh-。MIST分型将68株副溶血弧菌分为5个序列型(ST型),ST3为优势序列型(55/68),其余4种ST型中的ST189与ST345构成一个同源复合体CC345,且ST189为ST345的单位点变异型。结论 深圳地区副溶血弧菌所致的腹泻病暴发疫情,其流行菌株ST型别多样化,疾病控制中需警惕非主流ST型别引起暴发可能。     

Differing lifestyles of Staphylococcus epidermidis as revealed through Bayesian clustering of multilocus sequence types

Staphylococcus epidermidis is part of the normal bacterial flora of human skin and a leading cause of infections associated with indwelling medical devices. Previous phylogenetic analyses of subgenomic data have been unable to distinguish between S. epidermidis strains with nosocomial or commensal lifestyles, despite the identification of specific phenotypes and accessory genes that may contribute to such lifestyles. To attempt to better define the population structure of this species, the international S. epidermidis multilocus sequence typing database was analyzed with the Bayesian clustering programs STRUCTURE and BAPS. A total of six genetic clusters (GCs) were identified. A local population of S. epidermidis from clinical specimens was classified according to these six GCs, and further characterized for antibiotic susceptibilities, biofilm, and various genetic markers. GC5 was abundant and significantly enriched for isolates that were resistant to four classes of antibiotics, high biofilm production, and positive for the virulence markers icaA, IS256, and sesD/bhp, indicating its potential clinical relevance. In contrast, GC2 was rare and contained the only isolates positive for the putative commensal marker, fdh. GC1 and GC6 were abundant but not significantly associated with any of the examined characteristics, except for sesF/aap and GC6. GC3 was rare and identified as a potential genetic sink that received, but did not donate, core genetic material from other GCs. In conclusion, population genetics analyses were essential for identifying clusters of strains that may differ in their adaptation to nosocomial or commensal lifestyles. These results provide a new, population genetics framework for studying S. epidermidis.     

肺炎克雷伯菌脉冲场凝胶电泳及多位点序列分型技术的联合应用

目的 联合使用脉冲场凝胶电泳(PFGE)及多位点序列分型(MLST)对肺炎克雷伯菌进行分型研究.方法 参照美国疾病预防控制中心及亚太地区PulseNet提供的PFGE相关标准化操作程序,优化相关电泳条件,对某儿童医院于2005-2006年分离到的59株菌分型分析,对其中高度相似性菌株进行MLST分型分析.结果 59株菌经PFGE分型分析,可分成47个PFGE型,19个PFGE群,具有高度相似性的菌株经MLST分型,其型别分别为ST-340、ST-342、ST-343、ST-344、ST-345,其中ST-342、ST-343、ST-344、ST-345为MLST数据库中中国新发现的ST型.结论 具有PFGE高度相似性的肺炎克雷伯菌可以通过MLST进一步确认或区分.     

Population structure and genetic diversity of the parasite Trichomonas vaginalis in Bristol,UK

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, an extremely common, but non-life-threatening, sexually-transmitted disease throughout the world. Recent population genetics studies of T. vaginalis have detected high genetic diversity and revealed a two-type population structure, associated with phenotypic differences in sensitivity to metronidazole, the drug commonly used for treatment, and presence of T. vaginalis virus. There is currently a lack of data on UK isolates; most isolates examined to date are from the US. Here we used a recently described system for multilocus sequence typing (MLST) of T. vaginalis to study diversity of clinical isolates from Bristol, UK. We used MLST to characterise 23 clinical isolates of T. vaginalis collected from female patients during 2013. Seven housekeeping genes were PCR-amplified for each isolate and sequenced. The concatenated sequences were then compared with data from other MLST-characterised isolates available from http://tvaginalis.mlst.net/ to analyse the population structure and construct phylogenetic trees. Among the 23 isolates from the Bristol population of T. vaginalis, we found 23 polymorphic nucleotide sites, 25 different alleles and 19 sequence types (genotypes). Most isolates had a unique genotype, in agreement with the high levels of heterogeneity observed elsewhere in the world. A two-type population structure was evident from population genetic analysis and phylogenetic reconstruction split the isolates into two major clades. Tests for recombination in the Bristol population of T. vaginalis gave conflicting results, suggesting overall a clonal pattern of reproduction. We conclude that the Bristol population of T. vaginalis parasites conforms to the two-type population structure found in most other regions of the world. We found the MLST scheme to be an efficient genotyping method. The online MLST database provides a useful repository and resource that will prove invaluable in future studies linking the genetics of T. vaginalis with the clinical manifestation of trichomoniasis.     

多位点序列分型和脉冲场凝胶电泳在肠炎沙门菌分子分型的比较

目的 比较多位点序列分型技术和脉冲场凝胶电泳（PFGE）技术在肠炎沙门菌菌株间分型的分辨率。方法 分别建立肠炎沙门菌的6个管家基因thrA、pure、sucA、aroC、hemD、dnaN和一个特异性的DNA标记Sdf Ⅰ的多位点序列分型技术，及以寡切点的XbaⅠ、SpeⅠ作为限制性内切酶的PFGE方法，并应用上述方法对食品中的分离株进行分型，比较两种方法的分辨率。结果 PFGE可以将50株肠炎沙门菌分为11个型。并且通过双酶系统的双重PFGE分型，还可以将PFGE型别再精细划分为亚型；MLST则揭示了在同一血清型内部，各菌株之间的管家基因碱基序列高度保守，而在沙门菌不同血清型的核苷酸序列之间，则分别存在不同数量的碱基差异。即MLST方法只能用于血清型之间，不能在血清型内部进行分型研究。结论 与MLST比较，PFGE方法在肠炎沙门菌的分型方面显示了比较高的分辨率。     

单核细胞增生李斯特氏菌多位点序列分型

目的 了解河北省食源性单核细胞增生李斯特氏菌(Lm)的基因序列型(ST)分布特征,为Lm分子流行病学研究提供资料。方法 参考www.pasteur.fr/mlst网站建立的用于Lm分子分型检测的多位点序列分型方法(MLST),对分离自河北省的69株食源性Lm进行分型检测及分析,采用Start2和BURST对检测结果进行分析,绘制SplitsTree进化树。结果 69株Lm可分为16个基因序列型,辛普森指数(DI)为89.98%,发现新型ST705;河北省的优势ST型为ST8、ST87、ST2、ST1、ST3和ST9。结论 河北省优势的ST型型别相对较多,各ST型分布较分散,遗传具有多样性。     

多位点酶电泳法在新疆伤寒沙门氏菌分类和群体遗传学研究中的应用

采用多位点酶电泳（ＭＥＥ）法对新疆近１１年分离的１５４株伤寒沙门氏菌进行部分多位点酶基因型分类和群体遗传学研究。共测定分析７种代谢酶。研究结果表明,上述菌株共分为６９个电泳型（ＥＴ）,分属１０个克隆系（ＣＬａ～ＣＬｊ）,其中３０株爆发菌株分属２１个ＥＴｓ,３个ＣＬ（ＣＬｅ、ｆ和ｊ）,以ＥＴ１７、２０、２１、２４、２６和２７为主,占６１．３０％,ＣＬｆ为优势克隆系,菌株占９５．００％;７４株散发菌株     

纹带棒状杆菌多位点序列分型及应用

目的:建立纹带棒状杆菌的多位点序列分型（MLST）方法,探讨临床分离纹带棒状杆菌的种群结构和遗传进化关系。方法:筛选出7个管家基因（ gyrA、 gyrB、 hsp65、 sodA、 secA1、 rpoB、16S rRNA）,设计引物并进行PCR...     

Multilocus Sequence Analysis of Housekeeping Genes and Antigenic Determinant Genes in Bordetella pertussis Strains Isolated in Korea

Objectives

To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea.

Methods

Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST).

Results

More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999–2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI.

Conclusions

Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity.     

Clonal reproduction and linkage disequilibrium in diploids: a simulation study

Estimating the rate of clonal reproduction in natural population of diploid organisms is recognised as being problematic and even the detection of strictly clonal populations is often controversial. One well-acknowledged signature of clonal reproduction is the generation of non-random associations between loci. Linkage disequilibrium (LD) is thus often used for estimating the amount of clonal reproduction. Here we explore with computer simulations the effect of the rate of clonal reproduction on LD estimates obtained from different estimators within a comprehensive parameter range. None of the LD estimators studied is able to accurately measure the proportion of clonal (or sexual) reproduction on its own, due to strong bias, incoherent behaviour, or huge variances. The joint use of several statistics is thus recommended for the estimation rates of clonal reproduction in natural populations. We hope that our work will provide useful tools for the study of clonal diploids, many of which can only be studied with molecular markers, as it is the case for medically important parasites.     

广州市食品中蜡样芽胞杆菌生化分型和多位点测序分型分析

目的 了解广州市食品中蜡样芽胞杆菌的生化分型、多位点测序分型(multilocus sequence type,MLST)情况和优势序列型。方法 根据国标将79株食品来源蜡样芽孢杆菌进行生化分型,并根据PubMLST网站提供的蜡样芽胞杆菌MLST分型方案,扩增7个管家基因,经测序后,在PubMLST网站上比对获得菌株的序列型,利用eBURST V3和START2和MEGA6.0软件进行分析。结果 广州市食品中的蜡样芽胞杆菌优势序列型为ST26,占16.46%。结论 蜡样芽胞杆菌具有高度的遗传多样性。     

Recombination in viruses: Mechanisms,methods of study,and evolutionary consequences

Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure.