弗氏志贺菌4c和2a型菌株的脉冲场凝胶电泳分型特征

目的：了解北京部分地区弗氏志贺菌4c型（F4c）和2a型（F2a）菌株的分子分型特征。方法：对2005年和2006年自北京部分地区腹泻监测分离的弗氏志贺菌菌株（4c型10株,2a型20株）进行生化鉴定和血清分型,用PCR检测侵袭性抗原基因ipaH,用改良Kirby-Bauer纸片法检测菌株的耐药谱,用脉冲场凝胶电泳（PFGE）对菌株进行分子分型。结果：10株血清型鉴定为F4c的菌株中,有7株间的PFGE图谱存在相当的差异,Dice系数为0.78～0.92,而F2a菌株与大部分F4c菌株间的距离较远（Dice系数小于0.8）;F4c和F2a菌株对14种抗生素的耐药谱略呈差异。结论：采用PFGE能够很好地辨别弗氏志贺菌4c型和2a型菌株;弗氏志贺菌4c型菌株具有易变性,可在流行过程中产生PFGE图谱的差异、血清亚型的转换、耐药谱的变化等。     

空肠弯曲菌脉冲场凝胶电泳分子检测方法的建立及应用

摘要：【目的】建立空肠弯曲菌脉冲场电泳(pulsed-field gel electrophoresis, PFGE)图谱分型方法。【方法】在PFGE基本程序基础上,通过调整菌液浓度、Seakem Gold○R琼脂糖凝胶浓度、蛋白酶K浓度、洗涤方式和限制性内切酶SmaⅠ浓度,进行程序的比较与优化。应用PFGE技术对不同来源分离株进行分析。【结果】37株空肠弯曲菌脉冲场凝胶电泳图谱显示分离株均产生了6-24条电泳带,条带数量适中,清晰易读;系统进化树显示,可分为4个遗传谱系,分离株主要分布于PFGE遗传谱系     

大肠埃希菌耐药性及其基因同源性分析

目的 研究临床分离的大肠埃希菌对常用抗生索的耐药性及其基因分型,了解其耐药性趋势与传播流行情况,为临床合理治疗大肠埃希菌引起的感染提供参考依据。方法 采用常规鉴定技术鉴定细菌;采用K—B纸片扩散法测定77株大肠埃希菌对19种药物的耐药性;K—B法鉴定产超广谱β-内酰胺酶（ESBLs）;通过脉冲场凝胶电泳（PFGE）法对其进行基因分型以确定菌株之间的亲缘关系;FINGERPRINT Ⅱ软件进行细菌基因指纹图谱分析。结果 大肠埃希菌对青霉素类、喹诺酮类药物和氨曲南的耐药性明显增高,亚胺培南和美罗培南是大肠埃希菌感染患者的首选药物;经ESBLs确证试验,ESBLs阳性率为28．60％（22／77）;产ESBLs大肠埃希菌经PFGE指纹图谱分析,除第62株和第70株相似性系数为78．27％外,其余相似度均低于70．0％;ESBLs大肠埃希菌阴性株中除少数几对菌株相似性系数较高外,其余呈散在分布,且电泳带存有6条以上的不同条带,为流行病学无关的不同克隆。结论 大肠埃希菌对常用抗生索耐药性明显增高,且呈多重耐药趋势;该研究尚不能证明存在大肠埃希菌爆发性流行感染,提示可能存在院内感染大肠埃希菌的优势克隆;PFGE基因分型方法是耐药性与流行状况分析的有效手段。     

脉冲场凝胶电泳对铜绿假单胞菌的分子检测与相关性分析

目的应用PFGE对医院环境物体表面分离到的铜绿假单胞菌进行相关性检测与分析,探讨PFGE在监测和控制医院感染方面的意义。方法选择SpeⅠ酶对铜绿假单胞菌染色体DNA进行酶切,选用适宜的实验条件采用脉冲场凝胶电泳技术分析电泳酶切指纹图谱,了解其流行状况。结果14株铜绿假单胞菌基因组DNA经SpeⅠ限制性内切酶酶切后电泳,在30~800 kb之间产生18~24条大小不同的DNA切割片段区带。可分为9个带型。结论PFGE具有分辨力高,重复性好的特点,是铜绿假单胞菌分子流行病学分析的理想方法。     

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.     

2018年辽宁省沙门菌血清型、 分子分型及耐药性

目的 分析辽宁省2018年沙门菌血清型、脉冲场凝胶电泳(PFGE)及耐药特征。 方法 对2018年辽宁省分离到的35株沙门菌菌株进行鉴定后,采用脉冲场凝胶电泳及药物敏感试验对沙门菌进行分子分型及耐药性分析。 结果 经鉴定35株沙门菌分布于13种血清型,数量居前3位的血清型为肠炎沙门菌(37.14%)、鼠伤寒沙门菌(14.28%)和婴儿沙门菌(11.42%)。所有沙门菌菌株对萘啶酸和四环素耐药率较高,达到42.86%和34.28%,对头孢西丁和亚胺培南表现为不敏感。某些菌存在多重耐药性。 结论 2018年辽宁省沙门菌的耐药情况较为严重,血清型分布广泛,以肠炎沙门菌为主,绝大多数相同血清型的菌株与其PFGE条带聚集存在相关性。     

淋病奈瑟菌基因分型进展

淋病奈瑟菌表型分型方法的局限性限制其在该菌分型方面的应用。近年来,一系列该菌基因分型方法的建立弥补了表型分型方法的不足。各种基因分型方法,如Opa分型法、Por分型法、脉冲场凝胶电泳等有着各自不同的基因分型基础、应用范围及优缺点等。本文就近年来新发展的最主要的该基因分型方法作一简要介绍。     

2016-2019年辽宁省肠炎沙门菌分离株分子分型及其耐药性

目的 分析辽宁省肠炎沙门菌分离株的分子分型特征及耐药情况,为辽宁省肠炎沙门菌的分子流行病学及防控措施提供参考依据。 方法 采用PFGE分子分型方法对辽宁省2016-2019年肠炎沙门菌分离株进行分子分型,应用BioNumerics 7.6软件对酶切片段进行聚类分析,明确菌株的特征及同源性;采用最低抑菌浓度(MIC)法测定菌株对14种药物敏感性。 结果 共获得49株肠炎沙门菌,分子分型结果证明其呈17种PFGE带型,相似度区间为77.4%～100.0%,有2种优势带型;对萘啶酸的耐药率最高,达89.80%,其次氨苄西林的耐药率为69.39%,对3种以上抗生素的耐药率为55.10%。 结论 辽宁省肠炎沙门菌PFGE分子分型具有独特的优势带型,存在带型较多的特点;肠炎沙门菌分离株多重耐药状况比较严重,对萘啶酸的耐药率最高。     

食源性金黄色葡萄球菌脉冲场凝胶电泳分型分析

对石家庄地区食源性金黄色葡萄球菌进行脉冲场凝胶电泳(PFGE)分型,建立石家庄市金黄色葡萄球菌PFGE分型的数据库,探讨引起食物中毒金黄色葡萄球菌菌株和其他食源性金黄色葡萄球菌的分子特征。利用PFGE分型技术对2010至2012年石家庄市的75株食源性金黄色葡萄球菌进行全基因组分析,DNA酶切图谱用BioNumerics软件进行聚类分析。75株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在54%～100%之间,经聚类分析得到45个PFGE型别,6起食物中毒的金黄色葡萄球菌分别由PFGE01型、PFGE02型、PFGE11型、PFGE12型、PFGE15型和PFGE45型引起。建立的金黄色葡萄球菌PFGE分型数据库分子型别较多,且存在差异明显的多个克隆系,对石家庄地区金黄色葡萄球菌引起食物中毒的防控和溯源工作有重要意义。     

2014年大连市伤寒沙门菌耐药及 分子分型研究

摘要：目的 了解大连市伤寒沙门菌耐药性及分子分型特点,建立沙门菌分子特征本底信息,为今后防治工作提供科学依据。方法 采用微量肉汤稀释法对46株伤寒沙门菌进行8种抗生素敏感试验;运用脉冲场凝胶电泳（PFGE）方法对46株伤寒沙门菌进行分子分型及聚类分析。结果 46株伤寒沙门菌对萘啶酸（NAL）100%敏感,对氯霉素（CHL）和甲氧苄啶/磺胺甲噁唑（TMP/SMZ）耐药率为4.35%,对庆大霉素（GEN）耐药率为47.83%,发现多重耐药株1株;BioNumerics分析结果显示,46株伤寒沙门菌共产生30种PFGE带型,有7株表现为同一PFGE型别。结论 大连地区存在耐庆大霉素的伤寒沙门菌;PFGE结果表明这些菌株存在遗传多态性,并有优势菌株的存在。     

Antimicrobial susceptibility and pulsed-field gel electrophoresis of Shigella sonnei strains in Malaysia (1997-2000)

AIMS: Antimicrobial resistance of Shigella sonnei from Malaysia was determined and subtyping was carried by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these strains. METHODS AND RESULTS: A total of 62 isolates of S. sonnei from sporadic cases of shigellosis in different parts of Malaysia were studied by antimicrobial susceptibility test and PFGE. Approximately 35.5% of the strains showed resistance to three or more antimicrobial agents. Eight resistant phenotypes, i.e. RI to RVIII, was defined. Resistant phenotype RV and RVIII only appeared in year 2000. PFGE analysis with NotI and XbaI restriction showed that a great heterogeneity existed at the DNA level among Malaysian S. sonnei isolates. Fifty-eight NotI and 61 XbaI-PFGE profiles were observed in 63 S. sonnei isolates, including ATCC 11060 isolate. Drug sensitive isolates displayed very different profiles from drug-resistant isolates, with a few exceptions. Isolates of resistant phenotype RVI (SXTr.TETr.STRr) showed a greater similarity among each other compared with isolates of resistant phenotype RI and drug-sensitive isolates. CONCLUSION: Multi-drug-resistant S. sonnei were circulated in different parts of Malaysia and the emergence of new resistant phenotype was observed. Wide genetic variations among Malaysian S. sonnei were observed and the drug-sensitive strains could be differentiated from drug-resistant strains by PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study verifies the usefulness of PFGE in characterizing and comparing strains of S. sonnei. Minor variations among S. sonnei isolates could be detected by PFGE.     

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.     

Classification of Clostridium butyricum based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis

Eleven strains of Clostridium butyricum collected from different sources were analysed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pulsed-field gel electrophoresis (PFGE). The strains could be classified into four groups based on their banding profiles of the proteins extracted from the cells on SDS-PAGE. Group I consisted of seven strains, and these strains were further divided into five subgroups by PFGE. The strains belonging to groups II, III, and IV on SDS-PAGE were also classified into the same II to IV groups by PFGE. These data indicate that grouping of the strains of C. butyricum can be performed by employing both SDS-PAGE and PFGE.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

T-related Bacteriophages Isolated from Shigella sonnei

The properties of three T-related phages-35, 55, and 3201-isolated from Shigella sonnei were studied. They were similar with respect to morphology of plaques, duration of the latent periods, lysis inhibition effect, and serological characteristics. These phages closely resembled the T-even phages. Phages 3201, 35, and 55 had the same host range and receptor specificity as phage T2.     

Molecular typing by pulsed-field gel electrophoresis of Bacillus thuringiensis from root voles

The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE). For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes. Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B. thuringiensis from root voles is clonal. The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively. The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb. No significant differences were detected among the isolates subjected to biochemical properties determined by API tests. Present study showed that genomic diversity is a common feature of B. thuringiensis originating from one ecological niche. PFGE appears to be a useful technique for use in studies on the spread of B. thuringiensis in the environment.     

A cryptic plasmid from Shigella sonnei

pNZ500 is a 1.5 kb cryptic plasmid from a Shigella sonnei isolate. It was introduced into Escherichia coli by cotransformation, where it is maintained at about 30 copies per chromosome equivalent. Hybridization studies show that pNZ500 exhibits a high level of sequence similarity to other 1.5 kb plasmids found in different S. sonnei isolates but shares no homology with larger S. sonnei plasmids. pNZ500 shares a small degree of sequence homology with pBR322 and with pAC184. The homology with pBR322 is restricted to sequences close to the ori-bom region of this plasmid. Nevertheless, pNZ500 maintenance in E. coli is not dependent on DNA polymerase I activity, and does depend on continuing protein synthesis. pNZ500 encodes two polypeptide gene products whose monomer molecular weights are 24500 and 18000. The examination of host cells for the expression of possible plasmid phenotypes revealed no differences between cells bearing pNZ500 and plasmidless cells.     

Shigella sonnei Isolated from Well Water

A method is described which led to the isolation of Shigella sonnei from well water suspected of being the primary foci in a school-associated shigellosis outbreak.