产志贺样毒素和肠毒素大肠杆菌分子流行病学

[目的]人和动物腹泻的主要病原菌为大肠杆菌,本文主要研究贵州省致腹泻大肠杆菌毒力因子的分布类型.[方法]采用PCR技术对各毒力因子的基因分布进行研究.[结果]共分离到333株大肠杆菌,其中产肠毒素大肠杆菌(ETEC)在腹泻的人、猪、牛群中占优势,分别为:人群73(n=112),猪群82(n=106),牛群18(n=115).在ETEC菌株中检测到热敏肠毒素(lt)和不耐热肠毒素(st)基因,还存在lt/st并存现象.从人、猪、牛群中还检测到产志贺样毒素大肠杆菌(STEC),其中源自猪的STEC的检出率最高.大部分STEC同时携带lt、st或lt和st同时并存.编码F18菌毛的主亚基由fedA基因编码.对所分离大肠杆菌F18菌毛进行的研究结果表明,fedA基因主要与肠毒素基因共存,与stx基因并存的类型较少,25份猪源STEC菌株中仅有4份检测到fedA基因.[结论]贵州省人群、猪群和牛群致腹泻病原菌中以带F18菌毛的ETEC为主,STEC主要分布在腹泻的猪群中.     

奇异变形杆菌耐热性肠毒素的初步研究

为探讨奇异变形杆菌的发病机理,本文用乳鼠试验检测了从医院内腹泻患儿粪中分离的36株奇异变形杆菌肠毒素,结果4株呈阳性。对其中2株进行了耐热性试验,证明其具有耐热性,同时发现在人工培养条件下通过多次传代或肠毒素经反复冻融,耐热性肠毒素(ST)的活性可随之减弱或丧失。     

腹泻仔猪大肠埃希菌分离株ST4214的全基因组测序分析

目的 分析腹泻仔猪大肠埃希菌(E.coli)分离株血清型、毒力因子及耐药基因。方法 本课题组前期研究从腹泻仔猪粪便样本中分离到64株E.coli,本研究取其中含有毒力因子最多的1株进行全基因组测序分析。结果 E.coli分离株基因组序列为5.5 Mb,预测蛋白质编码序列数为5 743,与已知致病菌E.coli O145的基因重合率较低,为64.53%。经多位点序列分型分析,确定分离株为E.coli ST4214,血清型为O3∶H45。与病原菌O157∶H7、O145∶H28和O104∶H4的毒力基因比较表明：4株菌共同含有的毒力基因为354个,E.coli ST4214含有64种独特的毒力基因、编码鞭毛蛋白、Ⅳ型分泌蛋白、Ⅱ型分泌蛋白、溶血素、菌毛、肠毒素、荚膜多糖相关蛋白质。耐药基因E.coli分离株含有氨基香豆素抗性基因、磺胺类耐药基因、β-内酰胺抗性基因、多粘菌素耐药基因、肽抗生素抗性基因和抗生素耐药基因外排泵等多重耐药基因。结论 腹泻仔猪E.coli ST4214分离株是一株新菌株,具有较多毒力因子及耐药基因,可能与仔猪腹泻发病相关。     

大肠杆菌耐热性肠毒素STI三重串联突变体与黏附素K99融合基因的表达研究

产肠毒素大肠杆菌（ETEC）是一种导致仔畜和婴儿腹泻的主要病原之一,它的毒力因子主要有两类：黏附素（CFAs）和耐热性肠毒素（ST）或不耐热性肠毒素（LT）。通过PCR技术及双酶切连接技术,成功构建了含有3个STI突变体和1个黏附素K99基因的重组表达质粒pE3S(S)LK和pE3S(G)LK。重组菌株BL21(DE3) (pE3S(S)LK)和BL21(DE3)(pE3S(G)LK)的表达产物经SDS-PAGE和免疫印迹分析,表明以上两种重组菌株均能高效表达3STI(S)-K99和3STI(G)-K99融合蛋白,且融合蛋白能够被产肠毒素性大肠杆菌强毒株C83922 抗血清特异性识别。其次,利用乳鼠灌胃实验检测重组蛋白的生物学毒性,结果均为阴性(G/C值≤0.083),这表明该菌株已无STI生物学毒性。这些为研发预防大肠杆菌性腹泻的新型高效多价基因工程疫苗提供了基本素材和理论指导。     

2011年通州区腹泻源性嗜水气单胞菌的分子分型特征

目的了解北京市通州区2011年腹泻患者粪便中分离到的27株嗜水气单胞菌（Aeromonas hydrophila）的生物学和分子分型特征,为该菌引发疾病的防控提供参考依据。方法对27株腹泻源性嗜水气单胞菌进行Aer毒素检测和PFGE分型,并进行同源性比较。结果27株腹泻源性嗜水气单胞菌中7株菌的Aer毒素为阳性,占总数的25．93％;PFGE图谱分为27个带型。结论在通州区腹泻患者粪便中检出的菌株部分携带Aer毒力因子,目前无优势流行菌株。建议相关部门加强对该菌的监测,避免该菌引发的各类疾患的发生。     

人源产肠毒素大肠杆菌疫苗的研发进展

腹泻是全球范围内引起5岁以下幼童死亡的第二大病因,而产肠毒素大肠杆菌(ETEC)是引起腹泻的最常见病原菌,其产生的细菌定植因子(CFs)和肠毒素是关键的毒力因子。CFs介导细菌黏附宿主小肠上皮细胞并完成定植,产生热敏肠毒素(LT)和热稳定肠毒素(ST)破坏宿主上皮细胞内的体液平衡,使体液和电介质过量分泌从而导致腹泻。预防ETEC腹泻的首选方法是使用能激发宿主产生抗黏附素免疫力和抗肠毒素免疫力的疫苗,阻断ETEC黏附和定植并中和肠毒素。目前一种名为Dukoral~的霍乱疫苗因能刺激机体产生抗热敏毒素免疫,已经被一些国家批准用于短期保护和预防旅行者腹泻。新型试验性ETEC候选疫苗正在研发中,旨在提供保护期长、反应谱广的抗ETEC感染免疫保护力。本文针对疫苗研发的关键问题和研究现状作一综述,并对未来的研究作出展望。     

环境对产毒性大肠杆菌肠毒素表达的影响

通过体外试验,探讨一些与人体肠道微环境有关因素的变化对ＥＴＥＣ主要毒力因子肠毒素产生的影响。研究结果表明,细菌培养基的ｐＨ、渗透浓度、气体组成及温度的不同均可影响ＥＴＥＣ肠毒素的产生,而且ＬＴ与ＳＴ对有关环境因素的反应有所不同,一些与肠道环境有关环境条件的变化可影响ＥＴＥＣ肠毒素的表达,提示机体肠道微环境的变化可能与ＥＴＥＣ的致病有关。     

产肠毒素大肠杆菌亚单位疫苗3STaM(G)-K99和3STaM(S)-K99的纯化及其免疫学性质的研究

产肠毒素大肠杆菌(ETEC)是一种导致新生犊牛和仔猪腹泻的主要病原体之一.ETEC的毒力因子主要有黏附素(CFs)、不耐热性肠毒素(LT)和耐热性肠毒素(ST)三种.在前期研究中,利用PCR和酶切连接技术成功构建了两种ETEC亚单位疫苗3STaM (G)-K99和3STaM(S)-K99,且在大肠杆菌中获得高效表达.本研究利用阴离子交换层析纯化融合蛋白3STaM (G)-K99 and 3STaM(S)-K99,辅以弗氏佐剂免疫新西兰大白兔,通过Elisa分析其免疫学性质,并利用肠毒素中和实验在昆明系乳鼠中评价其激发抗STa中和抗体的能力.实验结果表明:亚单位疫苗3STaM(G)-K99 and 3STaM (S)-K99能够激发相对较高水平、可针对天然STa、ETEC和融合蛋白STa-K99的特异性抗体.其次,亚单位疫苗中STa突变体(STaM)组分的肠毒素活性显著降低,且其所激发的特异性抗体属于中和抗体,能有效抑制天然STa的肠毒素活性.亚单位疫苗3STaM (G)-K99 and 3STaM(S)-K99为研制预防ETEC感染性腹泻的多价基因工程疫苗提供了基本素材和理论指导.     

栗疫菌低毒力菌株dsRNA的分离及转移

自云南、广西、江苏、浙江、河北、京郊六个省市的板栗树(Castanea mollissima)溃疡斑上分离了栗疫菌(Endothia parasitica)160株,进行了dsRNA的提取及其毒力测定,获得两株含dsRNA的低毒力菌株(H),其毒力与带有起源于欧洲的dsRNA的低毒力菌株比较相近。比较国内菌株与欧洲低毒力菌株的dsRNA电泳谱带,示有共同分子量为5x106或6．2 x 108的电泳带。与正常毒力菌株(V)混合接种能显著降低致病力。这是欧美大陆发现低毒力菌株后,亚洲地区的首次报告。以5株具有欧洲dsRNA的低毒力菌株作为供体,与云南、江苏、浙江的10株毒力菌株配对,16％(8,50)的毒力菌株发生形态变化,dsRNA转入后毒力明显降低。结果表明,国内毒力菌株可以接受欧洲低毒力因子——dsRNA并转变为低毒力菌株。     

携带定居因子抗原的非毒素原性大肠杆菌 CF138 株特性的鉴定

对自腹泻病人粪便标本中分离的携带定居因子抗原的菌株（命名为ＣＦ１３８）的特性,侧重从两个方面进行了鉴定。通过电子显微镜观察,发现其菌体表面具有ＣＳ１及ＣＳ３纤毛结构,聚乙烯珠粘附试验结果证实具有粘附作用,甘露糖抗性血凝试验呈抗性凝集,与ＣＦＡ／Ⅱ抗血清作用显示强凝集阳性,属０６：Ｈ１６血清型,采用家兔肠攀试验,乳鼠ＳＴ活性测定及用ＬＴ、ＳＴ放射性ＤＮＡ探针检测等现行测定ＬＴ及ＳＴ肠毒素的方法,证实该株不产生肠毒素,结果表明该株属具有定居因子抗原的非毒素原性大肠杆菌自然诱变株。灌注该株菌体的免疫豚鼠产生了抵抗肠毒原性大肠杆菌攻击的保护力,表明该株系一潜在的抗ＥＴＥＣ感染的侯选菌苗株。     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Taxonomy of Citrobacter rods found in human specimens

The aim of this study was the identification of 181 Citrobacter strains on the basis of the recently proposed taxonomic changes of Brenner. All strains were isolated from diarrhoeic patients; 124 strains were originally sent for identification to Laboratory of Enterobacteriaceae DB NIH, 57 strains was isolated in Czech Republic. Citrobacter isolates were initially identified as C. koseri (3 strains), C. amalonaticus (1 strain) or as members of the C. freundii complex (177 strains). Additionally some biochemical tests were performed. The ability to grow in medium containing KCN, lysine decarboxylase production, lactose fermentation and PYR test were examined. Strains belonging to the C. freundii complex were identified to the species level by biochemical methods on the basis of the results of Brenner, who found some tests to be useful in separating Citrobacter species. These test included citrate and acetate utilization, arginine dihydrolase and ornithine decarboxylase activities, motility, urease production, esculin hydrolysis, and acid production from sucrose, dulcitol, melibiose, raffinose and salicin. On the basis of the criteria described above, 96.6% of the strains tested could be assigned to one of the recently named species of C. freundii complex. Using biochemical tests suggested by Brenner we were able to identify Citrobacter strains members of newly recognised species. A five-test system is proposed to identify the most frequently encountered species currently residing in the C. freundii complex.     

Enteroaggregative and cell-detaching<Emphasis Type="Italic">Escherichia coli</Emphasis> strains among polish children with and without diarrhea

To determine the association of enteroaggregative (EAEC) and cell-detaching (CDEC)Escherichia coli with diarrhea of unknown origin among children from Wroc?aw (Poland),E. coli strains isolated from stool specimens of children with diarrhea were examined for mannose-resistant adherence to HEp-2 cells. EAEC were isolated from 10 of 39 (26%) children examined with diarrhea and 4 of 20 (20%) age-matched controls. CDEC were present in 14 (36%) cases of diarrhea and 7 (35%) healthy subjects. Cell-detaching activity was distinctly associated with hemolysin production. Among hemolytic CDEC strains cytotoxic necrotizing factor 1 (CNF1) synthesis prevailed among isolates obtained from cases of diarrhea (57%) in comparison with isolates obtained from healthy controls (14.3%). Although neither EAEC nor CDECE. coli strains were associated with diarrhea of children in this setting, there were differences among EAEC and CDEC strains isolated from children with and without diarrhea.     

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.     

Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.)

AIMS: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. METHODS AND RESULTS: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42 degrees C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BI-phosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42 degrees C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76.9%) and Aerom. veronii (63.6%) were pathogenic for carp. CONCLUSIONS: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.     

醋酸钙-鲍曼不动杆菌复合体临床分离株多重PCR分型和感染特征分析

目的在临床微生物实验室建立醋酸钙-鲍曼不动杆菌复合体分型的方法,对相应菌株的药敏情况和临床特征进行分析,为临床准确诊断和有效治疗提供参考依据。方法收集苏州大学附属第一医院2015年10月至2016年1月临床分离的45株醋酸钙-鲍曼不动杆菌复合体菌株,用多重PCR法进行基因分型,用K-B法进行药敏试验,并采用SPSS 17.0软件进行统计分析。结果 45株醋酸钙-鲍曼不动杆菌复合体用多重PCR法鉴定为鲍曼不动杆菌38株,基因3型5株,基因13TU 2株,未鉴定出醋酸钙不动杆菌。标本来源以痰最多(35例),其次为血液标本(6例),脑脊液(2例)。科室分布在神经外科(11例)和ICU(10例)最多,其次是重症医学科(7例)和心胸大外科(6例)。年龄分布以41~60岁(17例)以及61~80岁(15例)两个年龄段最多;性别分布以男性较多(31例),女性较少(14例),而且各个年龄段男性均多于女性。在8种临床常用药物中,对头孢吡肟、哌拉西林、哌拉西林/他唑巴坦、环丙沙星和亚胺培南的耐药率均超过了70.0%,其中哌拉西林的耐药率最高(84.4%),阿米卡星的敏感率最高(51.1%)。结论多重PCR技术能对醋酸钙-鲍曼不动杆菌复合体进行快速基因分型,它成本低、操作简便,适合在临床微生物实验室推广。目前鲍曼不动杆菌引起的感染形势严峻,患者以中老年患者、男性、呼吸道感染、神经外科和ICU科室居多。鲍曼不动杆菌感染大多呈多重耐药甚至泛耐药。推荐临床通过积极治疗原发病,医护人员做好手卫生,加强消毒隔离,根据药敏结果合理选择抗生素等方法尽量减少和延缓耐药菌株的出现。     

Genomospecies identification and phylogenomic relevance of AFLP analysis of isolated and non-isolated strains of Frankia spp

Amplified fragment length polymorphism (AFLP) was tested as an alternative to the DNA-DNA hybridization technique (DDH) to delineate genomospecies and the phylogenetic structure within the genus Frankia. Forty Frankia strains, including representatives of seven DDH genomospecies, were typed in order to infer current genome mispairing (CGM) and evolutionary genomic distance (EGD). The constructed phylogeny revealed the presence of three main clusters corresponding to the previously identified host-infecting groups. In all instances, strains previously assigned to the same genomospecies were grouped in coherent clusters. A highly significant correlation was found between DDH values and CGM computed from AFLP data. The species definition threshold was found to range from 0.071 to 0.098 mismatches per site, according to host-infecting groups, presumably as a result of large genome size differences. Genomic distances allowed new Frankia strains to be assigned to nine genomospecies previously determined by DDH. The applicability of AFLP for the characterization of uncultured endophytic strains was tested on experimentally inoculated plants and then applied to Alnus incana and A. viridis field nodules hosting culture refractory spore-positive (Sp+, that sporulate in planta) strains. Only 1.3% of all AFLP fragments were shown to be generated by the contaminant plant DNA and did not interfere with accurate genomospecies identification of strains. When applied to field nodules, the procedure revealed that Alnus Sp+ strains were bona fide members of the Alnus-Myrica host infecting group. They displayed significant genomic divergence from genomospecies G1 of Alnus infecting strains (i.e. Frankia alni) and thus may belong to another subspecies or genomospecies.     

Appearance of enterotoxigenic Escherichia coli in piglets with diarrhea in connection with feed changes

Twelve weaned piglet (3-week-old) were divided into three groups according to time of feed change and observed for diarrhea during the time they were 3 to 8 weeks of age. A total of 553 strains of Escherichia coli were isolated from rectal fecal samples and examined for heat-labile (LT) and heat-stable (ST) enterotoxins, pilus antigens (K88, K99, and 987P), hemolysin (Hly), raffinose utilization (Raf) and drug resistance. Enterotoxins and/or hemolytic E. coli strains appeared in the rectal feces of 5- to 6-week-old piglets with diarrhea in connection with feed change and changing temperatures. Most of the isolates showed multiple drug resistance to sulfonamides (Sa), streptomycin (Sp), ampicillin, and/or mercury. Enterotoxigenic E. coli isolates represented four phenotypes: K88+.LT+.Hly+.Raf+.(SaSmCpTcKmSp) (12 strains), K99+.ST+.Raf+.(TcKm) (7 strains), ST+.Raf+.(TcKm) (7 strains), and ST.+(SaSmKm) (25 strains). The drug resistance determinants were transferable concurrently and some of them mobilized the determinants for K88, LT, Hly, and Raf to an E. coli C strain.     

Diarrhea caused by enterotoxigenic Bacteroides fragilis in children less than 5 years of age in Hanoi, Vietnam

Enterotoxigenic Bacteroides fragilis (ETBF) are considered as an emerging enteropathogen causing diarrhea in children. Eight hundred and thirty-six (836) children less than 5 years of age including 587 children with diarrhea and 249 age-matched controls were involved in the study. Within the group of children with diarrhea, 7.3% (43/587) ETBF was detected by immunoseparation in combination with polymerase chain reaction. The corresponding figure for the controls was 2.4% (6/249) (P<0.01). Within the diarrhea group, the prevalence was significantly higher in children older than 1 year of age. Three subtypes of ETBF isolates have been identified with the prevalence of 67.4%, 18.6%, and 16% for bft-1, bft-2, and a new bft, respectively. In the controls, two of the subtypes were identified, 5 bft-1 and 1 bft-2. More than half (55.8%) of the samples harboring ETBF also had other identified pathogens. The clinical symptoms of the single ETBF infection were not different from those of co-infections. This is the first study of the role of ETBF in children's diarrhea in Vietnam and it is concluded that this pathogen is an important causative agent of diarrhea in children in Hanoi, Vietnam.     

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.