多位点序列分型技术在沙门菌鉴定中的应用研究

目的:探讨多位点序列分型技术(Multilocus sequence typing,MLST)在沙门菌鉴定中的应用。方法:对1株分离自腹泻病人的沙门菌疑似菌株用肠杆菌科鉴定试条API 20E进行生化鉴定并进行血清学鉴定,应用MLST分型方法对该菌株分子分型。结果:API 20E鉴定为沙门菌属,Vi因子血清不凝集,O因子血清A-F O多价不凝集。该菌的MLST型别为ST64型,提示为鸭沙门菌。结论:MLST分子分型技术有助于沙门菌的鉴别。     

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。     

肠炎沙门菌多位点序列分型技术的研究

目的研究肠炎沙门菌菌株间的分子特征,建立了分子流行病学研究方法——多位点基因序列分型技术(MLST),并对食品中的肠炎沙门菌分离株进行分型研究。方法选择肠炎沙门菌的6个管家基因thrA、purE、sucA、aroC、hemD、dnaN及一个特异性的DNA标记SdfΙ作为目标基因。对上述基因分别进行PCR扩增及产物测序,用sequencer2.0软件对测序结果进行分析、比对核苷酸的差异。同时,将肠炎沙门菌株与GenBank中鼠伤寒沙门菌LT2相应的基因序列进行比较。结果在肠炎沙门菌标准菌株50041和18株食品分离株之间,6个基因PCR产物范围内的近60000个核苷酸序列完全相同,未观察到同一血清型内的基因突变。而与LT2相比,基因产物发生了1到6个点突变。在对SdfΙ的核苷酸序列比较研究中发现,肠炎沙门菌标准菌株与食品中分离株SdfΙ的核苷酸序列与GenBank中该序列的碱基对比,发生了C182T的点突变。即MLST技术揭示了在同一血清型内各菌株管家基因碱基序列的高度保守。结论MLST方法适用于不同血清型沙门菌株间的分型研究,而不适于同一血清型的菌株分型。     

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。     

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。     

杭州市2002-2008年甲型副伤寒沙门菌分子分型研究

目的研究2002-2008年杭州市甲型副伤寒疫情分离株的亲缘性,并探讨脉冲场凝胶电泳（PFGE）、多位点序列分型（MLST）在甲型副伤寒沙门菌分子分型中的应用。方法对404株甲型副伤寒沙门菌运用脉冲场凝胶电泳（PFGE）进行分子分型,并用K-B法测定菌株抗生素敏感性。挑选9株甲型副伤寒沙门菌进行多位点序列分型（MLST）以及稳定性试验。结果 404株甲型副伤寒沙门菌可分为6个PFGE型和4个耐药型,P1型和P2型属于同一个克隆系,该克隆系菌株占试验菌株数的99%,分离自临安市的310株菌均为P1型,分离自杭州市西湖区的主要菌型是P2型和P1型。9株甲型副伤寒沙门菌根据SucA位点的差异可分为2个MLST型。结论杭州市2002-2008年的甲型副伤寒疫情由同一克隆系的菌株主导。PFGE、MLST分型方法各有侧重,可互为补充。     

血流感染洋葱伯克霍尔德菌多位点序列分型研究

目的探讨血流感染洋葱伯克霍尔德菌的同源性,以便更好地控制短时间内洋葱伯克霍尔德菌引起的院内感染。方法对医院2013年5-6月分离的15株血流感染洋葱伯克霍尔德菌的菌株应用多位点序列分型方法(MLST),聚合酶链式反应(PCR)扩增atpD、gltB、gyrB、recA,lepA、phaC和trp B7个管家基因片段,测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型,研究其同源性。结果 15株菌中,13株菌的7个管家基因完全相同,同时gyrB管家基因出现新的序列型,为新的序列型(ST)。15株洋葱伯克霍尔德菌被分为3个基因型,分别为ST482、ST608和一个新的ST序列,来自神经外科的13株菌为同一个ST型。结论医院短时间内洋葱伯克霍尔德菌引起的血流感染属医院感染暴发,神经外科的13株菌为同一ST型。其中13株菌的ST型不仅相同且是新的ST型,与另外两科室的菌株不同。MLST在洋葱伯克霍尔德菌的分子生物学分型中的应用,表现出了较高的分辨率,对医院洋葱伯克霍尔德菌血流感染暴发的调查有重要意义。     

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.     

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

Objective To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. Methods Four selected different Eps, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease Xba Ⅰ , to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing Results By comparing the four results from each Eps, fk3 (switch time from 6s to 36s,total run time is 18.5 hours) seemed to be better than the others.59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340、ST-342、ST-343、ST-344、ST-345 by MLST. Among them, ST-342、 ST-343、 ST-344、 ST-345 types were all new MLST types that were reported in China.Conclusion Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.     

2006-2017年贵州省脑膜炎奈瑟菌多位点序列分型北大核心

目的了解2006-2017年贵州省脑膜炎奈瑟菌(Neisseria meningitidis,Nm)分离株的血清群和基因型特征。方法采用细菌分离培养和多位点序列分型(Multilocus sequence typing,MLST)技术,对2006-2017年贵州省Nm分离株进行血清群复核鉴定,对Nm的7个管家基因序列进行检测以确定克隆群(Clonal complex,CC)和序列型(Sequence type,ST),并构建遗传进化图谱以分析菌株间进化关系。结果共获得33株Nm,其中A群15株(45.5%)、C群4株(12.1%)、B群3株(9.1%)、W135群7株(21.2%)、不可分群4株(12.1%);得到6种CC和9种ST。A群、C群、W135群菌株的ST/CC基因型分别以ST-7/ST-5 Complex/subgroupⅢ(93.3%)、ST-4821/ST-4821 Complex(75.0%)、ST-6933/ST-174 Complex(85.7%)为主,B群基因型较分散。ST-5 Complex、ST-174 Complex基因型相对稳定,ST-4821 Complex具有遗传多样性;ST-5和ST-4821为高致病性克隆群。结论2006-2017年贵州省Nm存在多种基因型流行,A群、C群分别以ST-7/ST-5 Complex、ST-4821/ST-4821 Complex为优势基因型,B群基因型分散且活跃;需持续加强Nm分子流行病学监测。     

Phylogenetic analysis of carbapenem-resistant Acinetobacter baumannii isolated from different sources using Multilocus Sequence Typing Scheme

The emergence and worldwide distribution of carbapenem-resistant Acinetobacter baumannii strains has become a major public health threat. The objective of this study was to investigate the clonal relatedness of A. baumannii isolates collected from clinical and extra-hospital environments in Mthatha, South Africa. Forty carbapenem-resistant isolates comprising of clinical (20) and extra-hospital (20) were identified and tested for antimicrobial susceptibility. Detection of carbapenemase encoding genes was performed by Real-time PCR. The clonal relationship of clinical isolates relative to extra-hospital isolates was determined via multilocus sequence typing (MLST). All isolates (clinical and extra-hospital) were resistant to most common antibiotics including carbapenems (imipenem; MIC ≥32 μg/mL and meropenem; MIC ≥32 μg/mL) with the only exception being amikacin (with 3 isolates susceptible), tigecycline (14 isolates susceptible) and colistin (all isolates susceptible). The bla OXA-23-like and the intrinsic bla OXA-51 -like genes were detected in all the isolates tested. The bla OXA-58-like and bla IMP-type genes were detected in 2 clinical isolates whilst the bla OXA-24-like, bla VIM-type, bla NDM-1, bla SIM, and bla AmpC were not detected. The bla OXA-24-like, bla OXA-58-like, bla IMP-type, bla VIM-type, bla NDM-1, bla SIM, and bla AmpC were negative in the extra-hospital isolates. Co-occurrence of bla OXA-23 -like, bla OXA-58-like and bla IMP-type was observed in 2 clinical isolates. The MLST performed on 33 isolates identified 5 existing sequence types (ST) (ST1, ST2, ST25, ST85 and ST215) in clinical isolates and 2 existing STs (ST1 and ST2) in extra-hospital isolates. The most dominant ST was ST2 accounting for 68.8% of the clinical isolates and 82.4% of the extra-hospital isolates. The study demonstrated high prevalence and potential clonal spread of globally-disseminated clonal complex 2 carrying bla OXA-23-like within our local settings. However, ST25 might be an emerging lineage carrying the bla OXA-23-like . Continuous monitoring is important in limiting the spread of these strains in other healthcare settings and the community.     

沙门氏菌分离株自动化分型技术的应用研究

本研究尝试对沙门氏菌这一种常见致病菌进行自动化分型的研究,得出不同来源地沙门氏菌菌株的生化分型以及血清学分型数据,通过尝试建立沙门氏菌株分型信息为微生物污染的溯源提供一个可能的信息来源。运用VITEKR 2 Compact全自动微生物鉴定及药敏分析系统,对实验室分离的33株沙门氏菌进行分子分型研究,系统给出了21个生化编号,同时对以上菌种进行血清学分型研究,并将其分为16种血清型。     

葡萄球菌染色体mec盒多位点复合PCR分型测定

目的研究用多位点复合PCR分型法高效、快速、准确地测定金黄色葡萄球菌SCCmec型别。方法采用正交设计和平行对比分析,对影响测定结果的退火温度、延伸时间,MgCL2、dNTP、Taq DNA聚合酶、引物的用量进行了测试。结果获得多位点PCR测定的最佳退火温度为55℃、延伸时间为1 min,以及MgCl2、dNTP、TaqDNA聚合酶和各位点引物的最佳用量。结论多位点PCR测定是一种高效、快速、实用的SCCmec分型方法,可用于大规模的MRSA分子流行病学研究。     

Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis,Variable-Number Tandem-Repeat Analysis,Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing,and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods

Objectives

Mycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods.

Methods

Antibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed.

Results

All the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR.

Conclusion

The high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types.     

Development of a Multilocus Sequence Typing scheme for the study of Anaplasma marginale population structure over space and time

Bovine Anaplasmosis caused by Anaplasma marginale is a worldwide disease prevalent in tropical and subtropical regions where Rhipicephalus microplus is considered the most significant biological vector. Molecular markers previously applied for A. marginale typing are efficient for isolate discrimination but they are not a suitable tool for studying population structure and dynamics. Here we report the development of an MLST scheme based on the study of seven genes: dnaA, ftsZ, groEl, lipA, recA, secY and sucB. Five annotated genomes (Saint Maries, Florida, Mississippi, Puerto Rico and Virginia) and 53 bovine blood samples from different world regions were analyzed. High nucleotide diversity and a large proportion of synonymous substitutions, indicative of negative selection resulted from DnaSP 5.00.02 package application. Recombination events were detected in almost all genes, this evidence together with the coexistence of more than one A. marginale strain in the same sample might suggest the superinfection phenomena as a potential source of variation. The allelic profile analysis performed through GoeBURST shown two main CC that did not support geography. In addition, the AMOVA test confirmed the occurrence of at least two main genetically divergent groups. The composition of the emergent groups reflected the impact of both historical and environmental traits on A. marginale population structure. Finally, a web-based platform “Galaxy MLST-Pipeline” was developed to automate DNA sequence editing and data analysis that together with the Data Base are freely available to users.The A. marginale MLST scheme developed here is a valuable tool with a high discrimination power, besides PCR based strategies are still the better choice for epidemiological intracellular pathogens studies. Finally, the allelic profile describe herein would contribute to uncover the mechanisms in how intracellular pathogens challenge virulence paradigm.     

Campylobacter coli isolated in Brazil typed by core genome Multilocus Sequence Typing shows high genomic diversity in a global context

Campylobacter has been one of the most common causative agent of bacterial food-borne gastroenteritis in humans worldwide. However, in Brazil the campylobacteriosis has been a neglected disease and there is insufficient data to estimate the incidence of this pathogen in the country.AimsThe current study aimed to determine the phylogenetic relationships among Campylobacter coli strains isolated in Brazil and to compare them with international Campylobacter isolates available in some public databases.Methods and resultsA total of 63C. coli strains isolated in Brazil were studied. The MLST analysis showed 18 different STs including three STs not yet described in the PubMLST database. The cgMLST allocated the Brazilian strains studied into five main clusters and each cluster comprised groups of strains with nearly identical cgMLST profiles and with significant genetic distance observed among the distinct clusters. The comparison of the Brazilian strains with 3401 isolates from different countries showed a wide distribution of these strains isolated in this country.ConclusionsThe results showed a high similarity among some strains studied and a wide distribution of the Brazilian strains when compared to isolates from different countries, which is an interesting data set since it showed a high genetic diversity of these strains from Brazil in a global context. This study contributed for a better genomic characterization of C. coli strains isolated in Brazil and provided important information about the diversity of this clinically-relevant pathogen.     

脉冲场凝胶电泳技术在鼠伤寒沙门菌分型中的应用

[目的]探讨脉冲场凝胶电泳(PFGE)在鼠伤寒沙门菌分型中的应用。[方法]选择XbaⅠ酶对鼠伤寒沙门菌全基因组DNA进行酶切、脉冲场凝胶电泳。电泳带用Freeview软件进行相似性比较。[结果]13株鼠伤寒沙门菌经PFGE，根据其条带的差异可分为10个PFGE型别。[结论]PFGE法重复实验结果稳定，分型力较强，适合鼠伤寒沙门菌分型应用。     

一种伤寒沙门氏菌分型的新方法

目的 探讨伤寒沙门氏菌分型的新方法。方法 利用PCR扩增标记的Dig-dUTP-16SrRNA基因探针,分析40株伤寒沙门氏菌染色体经Pst Ⅰ消化后的16SrRNA基因限制性图谱。结果 40株伤寒沙门氏菌的分属14个不同的RT,各菌株杂交片段范围为4．4kb-29.7kb,每个RT内有杂交带5－10条不等,绝大多数为5－6条。结论 伤寒长期在某些地区流行可能与一些核糖核酸型仅出现这些地区,存在相对的地区性有关;同一个暴发点菌株具有相同的RT。16SrRNA基因限制性图谱分析可作为追踪传染源和探明传播途径的方法。