19群肺炎链球菌国家标准菌株分子特征分析及在菌株质量控制中的应用

目的明确19群肺炎链球菌(Streptococcus pneumoniae)国家标准菌株的分子特征,为完善中国肺炎链球菌国家标准菌株的质量标准提供依据。方法在传统肺炎链球菌菌种检定方法的基础上,应用16S rRNA基因分析、聚合酶链式反应(polymerase chain reaction,PCR)血清分型、多位点序列分型(multilocus sequence type,MLST)和脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型等多种分子生物学质控方法,对来源于中国医学细菌保藏管理中心的20株19群肺炎链球菌国家标准菌株进行分析。结果 20株19群肺炎链球菌的16S rRNA基因序列与肺炎链球菌模式株NCTC 7465的16S rRNA基因序列的相似性在99.64%～100%之间,碱基差异0～5 bp。PCR血清分型结果表明:11株19F型菌株均检测到大小为304 bp的19F型特异性扩增条带,6株19A型菌株均检测到大小为478 bp的19A型特异性扩增条带,PCR血清分型结果与血清凝集试验结果一致。MLST分型结果表明,相同血清型的菌株可以具有不同的序列型(sequence type,ST)。共获得12个ST型,其中6个ST型(10496、10497、10501、10502、10503和10509)为首次报道。PFGE分型结果显示:各型别菌株各具有其特征性PFGE带型(9～17条带),相同血清型或ST型菌株可能具有不同的PFGE带型。共存在18种不同的PFGE带型,其中19F型和19A型菌株中各有一对菌株的ST型和PFGE图谱完全一致。菌株的传代稳定性考察结果显示:在25代以内31708菌株的PFGE带型未发生变化,遗传特征是稳定的。结论 16S rRNA基因分析、PCR血清分型、MLST分型和PFGE分型等分子生物学方法应用于中国肺炎链球菌国家标准菌株的质量控制,可获得更加全面的肺炎链球菌标准菌株身份信息数据(包括序列、PCR扩增片段、序列型、图谱等),为进一步完善中国肺炎链球菌国家标准菌株的质量标准提供依据和数据支撑。     

沙门氏菌分型研究进展

沙门氏菌是重要的致病菌之一,该菌属型别繁多。沙门氏菌的分型方法可分为以表型特征为依据的表型分型方法和以基因特征为依据的分子分型方法。沙门氏菌的表型分型主要有血清分型和噬菌体分型。分子分型主要有分子血清分型、脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)、多位点序列分析(Multi-locus sequence typing,MLST)、多位点可变重复序列分析(Multi-locus variable numbers tandem repeat analysis,MLVA)以及成簇的规律间隔的短回文重复序列分析(Clustered regularly interspaced short palindromic repeats,CRISPR)等。与表型分型相比,分子分型技术具有快速、准确、重复性好等特点,现已得到广泛应用。其中,分子血清分型是鉴定沙门氏菌血清型的新方法,PFGE被认为是病原微生物分子分型的"金标准",MLST和MLVA以其分辨率高、可重复性好和可比性强等优势满足了全球流行病学发展的要求,能实现序列数据交换和共享,成为新一代的分子分型新工具,而近年发现的CRISPR分型对同源性较高的同种血清型具有较高的分型能力。但每种分型方法也有各自的优缺点和使用条件及适用范围,因此可以根据菌株特性、分型目的和实验室拥有的条件选择最合适的分型方法。     

无乳链球菌耐药性及分子分型方法的研究进展

无乳链球菌(Streptococcus agalactiae)是链球菌属最主要的致病菌之一,又被称为B群链球菌(group B Streptococcus,GBS)。S. agalactiae致病性主要由毒力因子和表面蛋白引起,毒力因子包括荚膜多糖、溶血素、菌毛岛屿、透明质酸酶、磷酸甘油激酶和CAMP因子,表面蛋白是αC蛋白、表面免疫相关蛋白、黏附蛋白、纤维蛋白原结合蛋白、层黏连蛋白结合蛋白和纤溶酶受体蛋白。近8年的S. agalactiae耐药情况统计数据发现,S.agalactiae已对19种抗菌抗药物产生耐药,检出20个耐药基因和12种毒力因子。国内外S. agalactiae分子分型方法主要致力于血清型、多位点序列、脉冲场凝胶电泳、菌毛岛屿和细菌前噬菌体基因分型。本文阐述了S.agalactiae生物学特性、流行性致病信息、耐药性研究现状和分子分型方法研究进展,以期为进一步探明S.agalactiae耐药机制、开发治疗S. agalactiae的新型药物提供参考。     

不同毒力差异基因型的新生隐球菌MLST分型及交配型鉴定研究

目的：了解及比较两组毒力差异明显的新生隐球菌格鲁比变种的多位点序列分型（MLST）的特点并进行交配型鉴定。方法采用多位点序列分型（MLST）的方法,设计7个看家基因（CAP59,GPD1,LAC1,PLB1,SOD1,URA5和IGS1）的引物,扩增并分析来源分别为环境和临床的各10株新生隐球菌格鲁比变种的基因型,并鉴定实验菌株交配型,与多位点微卫星分型（MLMT）结果对比,比较不同基因分型方法在分类中的稳定性和可靠性。结果在微卫星分型中为MLMT-36的10株环境分离株,MLST分型为ST-15,而在微卫星分型中为MLMT-13型的10株临床分离株,MLST分型为ST-32,所有菌株交配型均为MAT-α。结论MLST分型结果与MLMT分型结果高度一致,提示以上两种分子分型技术在真菌分类鉴定研究中可显示对于其分离背景及进化来源的高分辨率及稳定性。     

海南岛光滑假丝酵母菌多位点序列分型研究

目的:对海南省光滑假丝酵母菌临床分离株进行基因分型研究,了解菌株的遗传特征及遗传进化关系.方法:采用多位点序列分型(Multilocus sequence typing,MLST)技术,对临床分离的25株光滑假丝酵母菌6个管家基因序列进行测定;并将各个基因的序列与MLST数据库中储存的序列比对,确定其等位基因谱型及菌株序列型(STs).结果:25株临床分离的光滑假丝酵母菌通过MLST产生ST7、ST19、ST15、ST26、ST45共5个不同的序列型,其中ST7为主要序列型.结论:海南省光滑假丝酵母菌感染型别丰富,具有多样性;MLST分型具有较高的分辨力,可用于流行病学和菌群多态性的研究.     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

辽宁省食源性副溶血性弧菌PFGE分子分型及血清型、耐药谱

目的分析辽宁省副溶血性弧菌脉冲场凝胶电泳(PFGE)型别及血清型、耐药谱,探讨辽宁省副溶血性弧菌污染的同源性,为食源性疾病溯源和预警提供基础。方法对50株从辽宁省2015年食源性疾病和食品中分离的副溶血性弧菌进行PFGE分子分型、血清学分型、药物敏感试验,采用BioNumerics version 6.6软件进行分析,比较同源性。结果 50株副溶血性弧菌共分为19个血清型,食源性疾病分离株分为5个血清群,主要的血清型为O3群,其次为O1群;食品分离株分为9个血清群,主要的血清型为O3群,其次为O1和O4群。50株副溶血性弧菌对15种抗生素的耐药试验,对头孢唑啉耐药率最高达72%。50株副溶血性弧菌共分为4种PFGE优势带型,相似度为90.8%~100.0%。结论辽宁省食源性副溶血性弧的血清型以O3群和O1群为主,致病菌流行株血清型为O3:K6;食品中分离菌株血清型和PFGE带型非常分散,提示这些多为遗传不相关菌株。PFGE图谱可看出,绝大部分相同血清型菌株的PFGE带型相似度很高,O3与O1血清型菌株有高度同源性密切相关,耐药菌株间PFGE带型相似度很高,均具有高度同源性。相比2014年的耐药情况,对头孢唑啉耐药率显著增高。另外,少数菌株出现多重耐药,耐药情况不容乐观。     

基于多位点序列分型方法的于桥水库微囊藻遗传多样性分析

为了研究天津于桥水库微囊藻(Microcystis)的遗传多样性及其系统发育关系, 将分离自于桥水库的10株微囊藻的7个管家基因(ftsZ、glnA、gltX、gyrB、pgi、recA和tpi)构建了微囊藻的多位点序列分型(MLST)基因库, 与20个鄱阳湖序列型(STs)和237个日本湖泊的序列型进行比对, 结果发现于桥水库的STs呈独立分支, 说明相对于其他地区的藻株, 于桥水库微囊藻株具有共同祖先。MLST的结果显示本研究10个藻株具有较高的遗传多样性, 平均期望杂合度H值为0.938。基于序列类型构建的系统发育树显示, MLST可以有更精细的分辨率, 有成为种群分子标记的潜在可能性。     

肠道沙门菌O:4(B)菌群脉冲场凝胶电泳分子分型

为了明确2010年食源性疾病监测中分离的肠道沙门菌O:4(B)菌群的PFGE分子型别,探讨其多态性及其与分子流行病学关系,我们将分离获得的29株O:4(B)血清型沙门菌进一步鉴定培养,挑取单个菌落增菌,供试菌株基因组DNA用限制性内切酶Xba Ⅰ消化酶切后进行脉冲场凝胶电泳分型,所得结果用Bionumerics5.1软件进行聚类分析.实验结果表明,根据电泳指纹图谱,可将29株肠道沙门菌O:4(B)菌群分为24个PFGE型别,菌株间的相似值在56.31%~100%之间,同一血清型别的沙门菌有多个PFGE型别.脉冲场凝胶电泳对O:4(B)群沙门菌有较高的分型能力,可有效的应用于食物中沙门菌溯源分析及分子流行病学研究.     

分子分型技术用于流脑疫情的同源性分析

目的应用多位点序列分型(Multilocus sequence typing,MLST)技术和脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)技术对大连市2014年4月同一工地两起流脑疫情得到的脑膜炎奈瑟氏菌株进行分子分型图谱分析,了解各菌株之间的亲缘关系。方法对病例的脑脊液标本进行脑膜炎奈瑟菌PCR分群,对另一病例和所有密切接触者分离菌株进行多位点序列分型(MLST)试验和脉冲场凝胶电泳(PFGE)鉴定实验。结果疑似病例标本PCR结果为脑膜炎奈瑟氏菌A群阳性,另一病例和所有密切接触者分离到的菌株PFGE图谱基本相同,表明来自同一克隆系。多位点序列分析结果为ST7。结论两起疫情分离到的菌株型别之间具有高度的同源性,该病原菌类型为ST7的A群脑膜炎奈瑟氏菌。     

Epidemiological relationships of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from humans and chickens in South Korea

Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007–2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009–2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.     

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.     

Genetic diversity and antimicrobial resistance? in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.     

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.     

Clonal Analysis of Meningococci during a 26 Year Period Prior to the Introduction of Meningococcal Serogroup C Vaccines

Meningococcal disease remains a public health burden in the UK and elsewhere. Invasive Neisseria meningitidis, isolated in Scotland between 1972 and 1998, were characterised retrospectively to examine the serogroup and clonal structure of the circulating population. 2607 isolates causing invasive disease were available for serogroup and MLST analysis whilst 2517 were available for multilocus sequence typing (MLST) analysis only. Serogroup distribution changed from year to year but serogroups B and C were dominant throughout. Serogroup B was dominant throughout the 1970s and early 1980s until serogroup C became dominant during the mid-1980s. The increase in serogroup C was not associated with one particular sequence type (ST) but was associated with a number of STs, including ST-8, ST-11, ST-206 and ST-334. This is in contrast to the increase in serogroup C disease seen in the 1990s that was due to expansion of the ST-11 clonal complex. While there was considerable diversity among the isolates (309 different STs among the 2607 isolates), a large proportion of isolates (59.9%) were associated with only 10 STs. These data highlight meningococcal diversity over time and the need for ongoing surveillance during the introduction of new meningococcal vaccines.     

Multilocus Sequence Typing of Uropathogenic ESBL-Producing <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated in a Brazilian Community

In the present study, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction detection of three resistance genes were combined to characterize seven uropathogenic E. coli isolated from outpatients. Selected portions of seven housekeeping and three antibiotic-resistance genes of the isolates were sequenced. The seven isolates were classified into four different sequence types (STs) by MLST and five PGFE types. Three isolates had a novel allelic profile representing a new ST designated as ST528 and showed the same PFGE and resistance genes. Two isolates, both characterized as ST359, were differentiated by PFGE and shared only one of the antibiotic-resistance genes studied. Comparison of MLST results with those of PFGE and resistance genes demonstrated that Escherichia coli had acquired different antibiotic-resistance genes and DNA rearrangements, causing alterations in PFGE patterns but maintaining the same ST. Furthermore, this article also reports the first detection of a CTX-M-2 ESBL E. coli and SHV-5 in a Brazilian community.     

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.     

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae.