76株空肠弯曲菌血清学分型和药物敏感试验

空肠弯曲菌是引起人类急性腹泻，尤其是小儿腹泻的重要病原菌．近年来，作者[1]在调查本地区急性腹泻患者空肠弯曲菌感染情况的同时，对所分离的菌株进行血清学分型，借以对流行病学追溯空肠弯曲菌传染源和传播途径作一探讨，部分菌株还做了药物敏感试验．材料和方法一、菌株来源从济南地区急性腹泻病人及动物粪便分离菌株，其中人源31株，动物45株（鸡36株，猪7株，狗1株和猫1株）．全部菌株采取肛拭和新鲜粪便按王焕妞[2]方法鉴定．二、血清学分型被检菌经血平板分离纯化菌落传2～3代．挑取菌落，经鉴定具有触酶、氧化酶、醋酸铅纸条硫化…     

利用基因芯片技术分析空肠弯曲菌的遗传特征

目的 为分析我国弯曲菌遗传特征,本研究根据已发表多株弯曲菌的全基因组测序特征及比对结果自行设计基因芯片,利用芯片对我国不同宿主来源菌株进行遗传特异性分析。方法 根据前期基因组水平比对分析的结果,利用Combimatrix tilingCustomArrayTM 90K芯片,设计DNA芯片。芯片包含已测序菌株 ICDCCJ07001、269.97、NCTC11168、81-176、81-116和RM1221共3384个CDS的探针序列,以及空肠弯曲菌耐药及致病性相关2个质粒共80个CDS的探针序列,与脂寡糖的合成相关基因簇16种共219个CDS的探针序列、荚膜多糖合成相关基因簇7种共160个CDS的所有序列。对我国不同宿主来源27株分离菌株提取DNA,利用芯片进行杂交,获得杂交信息并分析不同菌株CDS分布特征分析及聚类特点。结果 中国菌株的主要变异区域主要存在于与脂寡糖、荚膜多糖合成相关的基因簇、鞭毛修饰相关的基因簇、DNA限制/修饰相关的基因簇以及空肠弯曲菌Mu样噬菌体基因簇。基因组水平不同来源菌株CDS分布的聚类结果没有发现显著的宿主归因特点,但GBS相关菌株脂寡糖合成相关基因组成具有共性特征。结论 通过验证以及与过去研究的比较,本次研究中的基因芯片技术结果准确可信,本研究所用基因芯片在分析空肠弯曲菌基因多态性方面具有很好的优势,可用于弯曲菌遗传特征和重要毒力因子的分析和检测。     

空肠弯曲菌耐药现状

空肠弯曲菌(Campylobacter jejuni,C.jejuni)是革兰阴性的,微需氧弯曲菌,是目前世界上发达国家和发展中国家普遍存在的主要食源性病原菌,也是旅行者腹泻的主要致病菌[1]。空肠弯曲菌的感染导致空肠弯曲菌病,其主要症状为腹泻性肠炎,此外研究证明空肠弯曲菌的感染与格林巴-利综合征(Guillain-Barre Syndrome,GBS)密切相关,是空肠弯曲菌感染造成主要疾病负担之一[2]。在自然界中,空肠弯曲菌广     

深圳地区不同来源空肠弯曲菌毒力基因分布及分子分型研究

目的 了解深圳地区市售禽类食品和腹泻病人中分离的空肠弯曲菌毒力基因分布及其分子分型情况。方法 根据特异性引物,运用聚合酶链式反应(PCR)检测空肠弯曲菌4种毒力基因(cdtB,cadF,flaA,virB11),应用脉冲场凝胶电泳分型技术(PFGE)对空肠弯曲菌分离株进行分子分型。结果 3种毒力基因在禽类食品分离株(5株)和腹泻病人分离株(5株)的分布情况一致,均携带cdtB和cadF,但均未检出virB11,两种来源菌株flaA的检出情况分别为3/5和2/5; PFGE聚类分析图谱显示,10株空肠弯曲菌经Sam I 酶切后可分别产生5～9条DNA条带的PFGE带型,其中食品株与病人株之间部分存在高度同源,同源性达到85%以上,但高度同源的菌株之间携带flaA毒力基因的分布不同。结论 深圳地区空肠弯曲菌存在cadF,cdtB,flaA等3种毒力基因,不同来源菌株之间同时具备多样性和同源性,提示空肠弯曲菌腹泻病人的感染来源可能与受污染的禽类食品密切相关,为该地区食源性空肠弯曲菌腹泻病提供基础性资料和依据。     

聚合酶链反应检测空肠和结肠弯曲菌的核酸

为了解急性腹泻病空肠和结肠弯曲菌的感染率，建立了空肠和结肠弯曲菌的ＰＣＲ检测方法。引物设计在结肠弯曲菌鞭毛Ａ基因高度保守区，的主增产物为４５０ｂｐ。     

实时荧光聚合酶链反应检测空肠弯曲菌

目的 探索一种快速、敏感、特异的空肠弯曲菌检验方法.方法 根据GenBank公布的空肠弯曲菌基因序列,在其保守区域设计特异性引物与探针以建立基于TaqMan探针的实时荧光聚合酶链反应(PCR)方法,并对方法的特异性、敏感性和重复性进行研究.结果 该方法对空肠弯曲菌可产生特异扩增信号,对其他非空肠弯曲菌菌属无响应,其检测敏感性可达10cfu/mL.反应体系具有很高的稳定性.结论实时荧光PCR可快速、特异、灵敏地检测空肠弯曲菌,具有较强的实际应用价值.     

McAb—Dot—ELISA对空肠弯曲菌快速鉴定的研究

应用本室研制的抗空肠弯曲菌共同抗原的单克隆抗体。以Dot-ELISA 对空肠弯曲菌进行快速鉴定.通过对实验条件的优选,确定了Dot-ELISA 的工作程序.对两种参考菌株(Pcnner.Loir)、人及10种动物来源的476株空肠弯曲菌进行了鉴定,结果均为阳性反应。可在3h 内报告结果.与11种其它细菌的试验结果表明,除与幽门弯曲茵发生弱阳性反应外,与另外其它10种细菌均为阴性反应.为空肠弯曲菌的鉴定提供了一种特异、快速的方法.     

空肠弯曲菌感染致人畜共患周围神经病的病原学分析

目的：寻找格林-巴利综合征(GBS)的病因及抗体检测。方法：对GBS患儿及其家庭成员和家禽家畜进行流行病学调查，分离培养大便中的空肠弯曲菌(CJ)和质粒分析及抗体测定。结果：10例GBS患儿中发现4例与CJ感染有关的人畜共患的周围神经病，分别在家禽家畜肢体瘫痪后1～3w出现GBS表现，大便培养CJ均为阴性，CJ抗体全部阳性；患者家禽家畜6份粪便CJ培养有5例阳性，血清抗体均阳性。结论：空肠弯曲菌感染可引起格林-巴利综合征，防止CJ感染是降低GBS发生的重要措施。     

Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide

A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been cloned from gonococcal strain F62. The locus contains five open reading frames. The first and second reading frames are homologous, but not identical, to the fourth and fifth reading frames, respectively. Interposed is an additional reading frame which has distant homology to the Escherichia coli rfaI and rfaI genes, both glucosyl transferases involved in lipopolysaccharide core biosynthesis. The second and fifth reading frames show strong homology to the lex-1 or lic2A gene of Haemophilus influenzae, but do not contain the CAAT repeats found in this gene. Deletions of each of these five genes, of combinations of genes, and of the entire locus were constructed and introduced into parental gonococcal strain F62 by transformation. The LOS phenotypes were then analyzed by SDS-PAGE and reactivity with monoclonal antibodies. Analysis of the gonococcal mutants indicates that four of these genes are the glycosyl transferases that add GalNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1--4 to the substrate Glc beta 1-->4Hep--R of the inner core region. The gene with homology to E. coli rfaI/rfaI is involved with the addition of the alpha-linked galactose residue in the biosynthesis of the alternative LOS structure Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->4Hep-->R. Since these genes encode LOS glycosyl transferases they have been named lgtA, lgtB, lgtC, lgtD, and lgtE. The DNA sequence analysis revealed that lgtA, lgtC, and lgtD contained poly-G tracts, which, in strain F62 were, respectively, 17, 10, and 11 bp. Thus, three of the LOS biosynthetic enzymes are potentially susceptible to premature termination by reading frame changes. It is likely that these structural features are responsible for the high-frequency genetic variation of gonococcal LOS.     

检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用

目的 建立一种检测胎儿弯曲菌胎儿亚种、性病亚种和龟亚种的多重PCR检测方法。方法 使用针对胎儿弯曲菌、性病亚种和龟亚种的特异性引物,优化反应体系及反应条件,使用38株菌株（18株胎儿弯曲菌和20株非胎儿弯曲菌）验证其特异性,并将该方法应用于53份爬行动物粪便筛查。结果 针对3种亚种均可扩增相应的片段,胎儿亚种只有1条359 bp大小的条带、性病亚种有2条分别为359 bp和156 bp大小的条带,龟亚种有2条分别为359 bp和266 bp大小的条带。优化后的最佳扩增条件：退火温度58℃,引物MG3f和Cf359r浓度为0.1μmol/L,ISC2mf和ISC2mr、CoA266f和CoA266r的浓度0.2μmol/L。对胎儿亚种、性病亚种和龟亚种的检出限分别为0.40 ng/μL、0.39 ng/μL和0.47 ng/μL。使用18株胎儿弯曲菌和20株非胎儿弯曲菌其特异性为100%。对53份爬行动物粪便进行筛查中有1份龟亚种阳性并分离出了相应的菌株。结论 本研究建立的多重PCR方法能利用1个反应体系同时鉴别3种亚种,从而为菌株快速鉴定、流行病学调查和溯源研究提供技术支持。     

Genetic recombination as a major cause of mutagenesis in the human globin gene clusters

ObjectivesHomologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the α- and β-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere.ConclusionHere, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.     

百日咳杆菌百日咳毒素基因遗传变异分析

目的 了解百日咳毒素(pt)基因启动子区及不同亚基编码区遗传变异特点及进化规律,为分子流行病学监控和疫苗研究提供参考依据.方法 从GenBank数据库中下载已公开发表的百日咳菌株pt基因启动子区序列及S1～S5亚基编码序列,利用PhyML 3.0软件构建系统发育树,确定进化簇;利用MEGA 7.0软件计算遗传距离;通过...     

外排泵基因Rv1456c、Rv1457c、Rv1458c表达与结核分枝杆菌耐药关系探讨

目的研究外排泵基因Rv1456c、Rv1457c、Rv1458c的表达与结核分枝杆菌（MTB）不同耐药表型的关系。方法提取2017 — 2018年浙江省宁波市疾病预防控制中心耐药监测期间收集的102株MTB核糖核酸（RNA），应用实时荧光定量PCR方法检测ABC转运蛋白超家族外排泵基因Rv1456c、Rv1457c、Rv1458c的表达量，采用Mann-Whitney U检验分析外排泵基因表达在不同耐药表型菌株中的差异。结果Rv1457c和Rv1458c基因表达量在利福平耐药组中的中位数及四分位数间距［0.507（0.378 ~ 1.444），1.842（1.325 ~ 2.628）］高于利福平敏感组［0.418（0.357 ~ 0.618），1.545 （1.189 ~ 2.065）］，差异有统计学意义（Z＝2.030、2.108，均P＜0.05）。 耐多药组Rv1457c和Rv1458c基因表达量的中位数及四分位数间距［0.538（0.419 ~ 1.490），1.941（1.471 ~ 2.659）］，高于非耐多药组［0.415 （0.337 ~ 0.618），1.533 （1.122 ~ 2.056）］，差异有统计学意义（Z＝2.865、2.896，均P＜0.05）。 异烟肼耐药组和敏感组中Rv1456c、Rv1457c的高表达率均存在差异（χ2＝4.858、6.789，均P＜0.05），利福平耐药组和敏感组中Rv1456c的高表达率差异有统计学意义（χ2＝8.424，P＜0.05），链霉素耐药组和敏感组中Rv1457c的高表达率差异有统计学意义（χ2＝4.545，P＜0.05），乙胺丁醇耐药组和敏感组中Rv1456c、Rv1457c的高表达率差异均有统计学意义（χ2＝5.142、10.202，均P＜0.05）。结论外排泵基因Rv1457c、Rv1458c的相对表达量增加与MTB对利福平耐药有关。     

Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae

The aim of the present study was to examine the stability and evolution of tet(M)-mediated resistance to tetracyclines among members of different clonal lineages of Streptococcus pneumoniae. Thirty-two tetracycline-resistant isolates representing three national (Spanish serotype 14, Spanish serotype 15, and Polish serotype 23F) and one international (Spanish serotype 23F) multidrug-resistant epidemic clones were all found to be tet(M) positive and tet(O), tet(K), and tet(L) negative. These isolates all carried the integrase gene, int, which is associated with the Tn1545-Tn916 family of conjugative transposons. High-resolution restriction analysis of tet(M) products identified six alleles, tet(M)1 to tet(M)6: tet(M)1 to tet(M)3 and tet(M)5 in isolates of the Spanish serotype 14 clone, tet(M)4 in both the Spanish serotype 15 and 23F clones, and tet(M)6, the most divergent allele, in the Polish 23F clone. This indicates that tet(M) variation can occur at the inter- and intraclone levels in pneumococci. Two alleles of int were identified, with int1 being found in all isolates apart from members of the international Spanish 23F clone, which carried int2. Susceptibility to tetracycline, doxycycline, and minocycline was evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracyclines. Resistance to tetracyclines was found to be inducible in isolates of all clones; however, the strongest induction was observed in the Spanish serotype 15 and 23F clones carrying tet(M)4. Tetracycline was found to be the strongest inducer of resistance, and minocycline was found to be the weakest inducer of resistance.     

Genetic localization and molecular characterization of the nonS gene required for macrotetrolide biosynthesis in Streptomyces griseus DSM40695

The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (+/-)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (+/-)-NA suggests that NonS catalyzes the formation of (-)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.     

O1和O139群霍乱弧菌超级整合子VchintIA基因的变异

目的 研究不同血清群(型)霍乱弧菌超级整合子整合酶基因(VchintIA)的变异及其与霍乱弧菌分类的关系。 方法 使用聚合酶链反应(PCR)和序列测定的方法对VchintIA基因进行扩增和序列分析。 结果 在实验菌株中,O1El Tor和O139群霍乱弧菌产毒株以及毒素共调菌毛(toxin co-regulated pilus, tcp)基因簇阳性的O1群非产毒株的VchintIA序列完全一致,tcp基因簇阴性的O1群非产毒株的VchintIA序列在核苷酸水平上与产毒株不同,但是在氨基酸水平上相同。O139非产毒株的VchintIA序列在核苷酸和氨基酸水平上均与产毒株的VchintIA不同。 结论 O1和O139霍乱弧菌超级整合子VchintIA存在核苷酸和氨基酸水平上的变异,O139非产毒株中VchintIA的变异与其整合子内部的结构可能相关。