福氏2a志贺菌肠毒素ShET1/ShET2基因分型在菌痢快速诊断中的应用

目的对福氏2a志贺菌肠毒素ShET1(Shigellaenterotoxin1)和肠毒素ShET2(Shigellaenterotoxin2)进行基因分型,提高菌痢爆发流行时同源克隆的鉴定分析水平。方法对93株分离自不同地区,不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1/ShET2基因,进行基因分型和同源克隆鉴定。结果93株福氏2a志贺菌按志贺菌肠毒素ShET1/ShET2基因可分为4种基因型,即12株ShET1(-)/ShET2(+),14株ShET1(+)/ShET2(-),59株ShET1(+)/ShET2(+),8株ShET1(-)/ShET2(-)。93株福氏2a志贺菌ShET1检出率为89.24%(83/93),ShET2为65.59%(61/93)。二者至少有一种基因被检出的检出率为91.39%(85/93)。结论福氏2a志贺菌的快速诊断可应用ShET1、ShET2双基因PCR检测,具有较高的敏感性与特异性。在应用多生物学标志进行福氏2a志贺菌同源克隆鉴定系统研究时,肠毒素ShET1/ShET2基因PCR分析是不可或缺的分析指标。     

志贺菌属肠毒素ShET1/ShET2的基因型PCR分析

目的 对福氏2a志贺菌肠毒素ShET1和肠毒素ShET2进行基因分型，提高福氏2a志贺菌同源克隆鉴定系统的分析水平。方法 对93株分离自不同地区、不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1／ShET2基因并进行基因分型。结果 93株福氏2a志贺菌按志贺菌肠毒素ShET1／ShET2基因分为4种基因型，即12株ShET1(-)／ShET2( )，14株ShET1( )／ShET2(-)，59株ShET1( )／ShET2( )，8株ShET1(-)／ShET2(-)。结论 福氏2a志贺菌肠毒素ShET1／ShET2基因PCR检测可用于福氏2a志贺菌的初步筛检。     

基因探针和PCR方法在菌痢流行病学研究中的应用

目的 探讨基因探针和PCR方法在F2a志贺氏菌痢暴发的流行病学研究中的意义。方法 对43株自患者粪便和食物中分离到的F2a志贺氏菌进行16srRNA、ipaH基因Southern杂交，随机PCR和set1／set2毒力基因PCR分析。结果 随机PCR、ipaH基因Southern杂交将43株F2a志贺氏菌分为两个不同的基因型，set1／set2毒力基因PCR分为三个不同的基因型，16srRNA基因Southern杂交均为同一基因型。结论 基因分型方法能更准确、深入地揭示菌痢暴发过程中各分离株之间的流行病学联系。其中set1／set2毒力基因PCR方法具有较高的分辨率，在F2a志贺氏菌的分子流行病学研究中具有重要的作用。     

福氏志贺菌中质粒介导喹诺酮类耐药qnr基因的检测

目的 了解福氏志贺菌中qnr基因的分布.方法 采用PCR法检测26株福氏志贺菌的qnr基因并对阳性扩增产物进行测序分析,采用琼脂稀释法测定qnr基因阳性菌株对多种抗菌药物的敏感性,采用随机引物PCR法(ERIC-PCR)进行qnr基因阳性菌株同源性检测.结果 26株福氏志贺菌中,qnrAl、qnrS1和qnrS2基因的阳性率分别为30.8%、11.5%和3.8%;qnr基因阳性菌株对氯霉素、奈啶酸耐药.对诺氟沙星、哌拉西林耐药或中介;部分菌株对头孢唑啉、阿米卡星、第二、三代头孢菌素耐药.ERIC-PCR电泳图谱型显示.2株qnrAl基因阳性菌株属于同一谱型.结论 福氏志贺菌中存在qnr基因阳性菌株,部分菌株间存在克隆传播现象,临床应加强检测和监测.     

福氏志贺菌4av和Yv血清型PCR鉴定方法的建立及应用

目的 建立福氏志贺菌4av和Yv血清型PCR鉴定方法。方法 根据福氏志贺菌4av和Yv血清型O抗结构,针对O抗合成基因wzx、IV型抗原决定基因gtrIV和MASF IV-1抗原决定基因opt,建立血清型4av和Yv 的PCR鉴定方法。并应用该方法对126株福氏志贺菌临床分离株进行血清型检测。结果 建立了一种福氏志贺菌4av和Yv血清型PCR鉴定方法,在一个反应中包括4对引物,Yv血清型PCR扩增为wzx及opt阳性;4av血清型PCR扩增为wzx、opt及gtrIV阳性。该方法可将4av和Yv血清型与目前已知的其他福氏志贺菌血清型完全区分。对126株不同血清型福氏志贺菌临床分离株的鉴定结果显示,该方法具有很好的特异性。结论 本研究建立的福氏志贺菌4av和Yv血清型PCR鉴定方法,可以用于志贺菌检测和监测。     

福氏2a志贺菌痢暴发的RAPD分析

目的 探讨江门一起菌痢暴发的特异传染源。方法 对8株福氏2a志贺菌进行PAPD分析。结果 此次菌株暴发期间分离的8株福氏2a志贺菌具有两种不同的RAPD特征（RTI、RTⅡ型）,其中2株病例密切接触者分离株J98101(PTⅡ型）和J9826（RT I型）分离不同克隆。结论 本次暴发偶合有部分散发病例。相对于质粒分析而言,RAPD分析具有更高的分辨率,它提供的遗传学信息更为丰富、直接、精细。     

福氏志贺菌主动外排基因acrA的克隆及原核表达

目的对福氏志贺菌外排基因acrA进行克隆和原核表达,为进一步研究其在志贺菌外排机制中的作用奠定基础。方法参考福氏志贺菌2aacrA基因序列设计一对特异引物,在引物的5′和3′端分别加入含有BamHⅠ和SalⅠ限制性酶切位点序列。以福氏志贺菌2a菌株基因组DNA为模板,通过PCR扩增acrA基因并与pMD18-T载体连接,然后转化DH5α。提取重组质粒pMD18-acrA,经BamHI/SalI双酶切并与载体pET30a连接后转化宿主菌BL21(DE3)pLys,通过IPTG诱导表达目的蛋白。结果克隆的acrA基因长度为1122bp,核苷酸序列与GenBank上公布的序列完全相同。原核表达经SDS-PAGE及WesternBlotting检测和鉴定,结果表明重组载体pET-30a-acrA可成功地在大肠杆菌中表达AcrA蛋白。结论成功构建了福氏志贺菌acrA基因的原核表达质粒,并在大肠杆菌中得到有效表达,该研究为了解AcrA蛋白的特性、功能以及对福氏志贺菌多药耐药机理的深入研究奠定了基础。     

福氏志贺菌对诺氟沙星耐药性及耐药机制的研究

目的研究福氏志贺菌对诺氟沙星的耐药性及耐药机制。方法收集福氏志贺菌３０株,进行抗菌药物敏感试验,４株耐药菌,１株敏感菌和福氏志贺菌标准菌５１５７３的ｇｙｒＡ基因的Ｎ末端编码区进行ＰＣＲ扩增并克隆和测序。结果福氏志贺菌对诺氟沙星的耐药率高达５３．３％,ｇｙｒＡ基因的序列分析结果揭示存在３个导致氨基酸改变的基因点突变：丝氨酸８３→亮氨酸,天冬氨酸８７→天冬酰氨,天冬氨酸８７→甘氨酸。结论福氏志贺菌对诺氟沙星已有较高的耐药性,ｇｙｒＡ基因点突变与福氏志贺菌对诺氟沙星的耐药性有关。     

福氏志贺菌PFGE和MLVA分子分型方法的应用与评价

目的 比较和评价脉冲场凝胶电泳技术(PFGE)和多位点可变数目串联重复序列分析技术(MLVA)在福建地区福氏志贺菌研究中的应用。方法 运用PFGE和MLVA技术对43株分离自福建地区的福氏志贺菌进行分子分型,结合流行病学资料分析比较分型效果。结果 43株福氏志贺菌经PFGE分型后相似度在61.70%~100%之间,按照100%的相似水平可分为36个PFGE型,没有优势PFGE型别,分辨系数为0.992 2,存在4个优势簇(G1~G4);福氏志贺菌经MLVA分型后,按照100%的相似水平可分为41个MLVA型别,遗传关联度介于6.207％～100％之间,没有优势MLVA型别,分辨系数为0.997 8,得到5个优势基因群(Cluster1~5)。在最小生成树上,部分F4c与Fx亲缘关系较近,并都由F2a和F1a分支而来,表现出一定的遗传关系。结论 两种分型方法的分辨率基本一致,PFGE分型结果与流行病学背景资料及血清型别基本吻合,MLVA在分析菌株种群进化关系上更具优势。     

2007年中国四省福氏志贺菌分离菌株的毒力基因检测和PFGE分析

目的分析中国河南等四省的福氏志贺菌分离菌株毒力基因和脉冲场凝胶电泳(PFGE)分型。方法应用PCR方法检测2007年从河南、青海、甘肃和山西分离的262株福氏志贺菌的侵袭性质粒抗原H基因(ipaH)、志贺肠毒素2基因(sen)、志贺肠毒素1基因(set1A)以及侵袭性蛋白基因(ipaBCD)。参考美国CDC的PulseNet实验方法 ,用限制性内切酶NotI对细菌染色体进行酶切,对这些分离菌株进行PFGE分析,使用BioNumerics软件进行聚类分析,并按照PulseNet命名原则对带型进行命名。结果 262株福氏志贺菌分离菌株ipaH、sen、set1A和ipaBCD基因的携带率分别为100%、93.89%、96.18%和92.75%。具有7种毒力基因携带模式,其中89.31%的菌株为Ⅰ型毒力基因携带模式(ipaH+sen+set1A+ipaB-CD+),有99.24%菌株同时携带两种或以上毒力基因。262株福氏志贺菌共分为83个PFGE型别,其中CNJZXN11.0002、CNJZXN11.0003、CNJZXN11.0060、CNJZXN11.0081、CNJZXN11.0137、CNJZXN11.0169、CNJZXN11.0190、CNJZXN11.0195、CNJZXN11.0196、CNJZXN11.0199、CNJZXN11.0217、CNJZXN11.0325和CNJZXN11.0327为13种主要带型。CNJZXN11.0003型菌株在4省均有分布,CNJZXN11.0199为河南菌株的优势带型,CNJZXN11.0137、CNJZXN11.0325和CNJZXN11.0327为山西特有的PFGE带型,与CNJZXN11.0003等主要带型聚类相似性较小。结论四省分离的福氏志贺菌株中ipaH、sen、set1A和iPaBCD毒力基因的携带率较高,而且这些菌株的PFGE型别既具有地区性差异,又具有优势型别的交叉。     

Distribution of Shigella enterotoxin genes and secreted autotransporter toxin gene among diverse species and serotypes of shigella isolated from Andaman Islands, India

We studied the prevalence and distribution of the newly described genes for Shigella enterotoxins (ShET1 and ShET2, encoded by set and sen genes) and secreted auto-transporter toxin (encoded by sat gene) in clinical isolates from the Andaman Islands, India. A total of 153 Shigella isolates obtained from hospitalized patients during 1994-2004 were analysed. These isolates included all the four species of Shigella (S. dyseteriae-29, S. flexneri-75, S. sonnei-38, S. boydii-5) that belonged to diverse serotypes (including serologically untypable-6) and each serotype included a wide variety of genotypes. Each isolate underwent polymerase chain reaction (PCR) for detection of set, sen and sat genes employing specific primers. We found the set gene in all S. flexneri 2a and 2b isolates (41 of 41, 100%) but not outside S. flexneri serotype 2. The sen gene was well distributed among all species and serotypes but its presence was apparently low at 49.1% (75 of 153), probably because of the loss of the large plasmid that harbours the gene in 76 of the 78 (97.4%) sen negative isolates. Also, all S. flexneri 2 isolates (including 2a and 2b serotypes) had the sat gene. It was present in 96% (72 of 75) of S. flexneri, in 6.9% (2 of 29) of S. dysenteriae, in 20% (1 of 5) of S. boydii, and in 33.3% (2 of 6) of untypable Shigella, but not in (0 of 38) S. sonnei. This study provides initial data on the prevalence and distribution of of the set, sen and sat genes in a wide variety of Shigella isolated over a 10-year period. Our results suggest a greater prevalence of the set and sat genes in S. flexneri 2 isolates than previously thought and might help in future pathochip designs.     

Effect of shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo.

BACKGROUND: Shigella enterotoxin 1 is a novel enterotoxin elaborated by Shigella flexneri 2a that causes fluid accumulation in rabbit ileal loops and a rise in short circuit current in Ussing chambers. AIMS: To gain insights into the mechanism of action of shigella enterotoxin 1. METHODS: Supernatants from genetically engineered clones either overexpressing shigella enterotoxin 1 or producing deletion mutants of the toxin were tested in rabbit ileum both in vitro and in vivo. RESULTS: In rabbit ileum shigella enterotoxin 1 induced an irreversible rise in short circuit current that was not mediated by any of the recognised intracellular mediators of secretion. Deletion of 90% of the A subunit of the holotoxin ablated its enterotoxicity. In the in vivo perfusion model, the toxin induced a time dependent decrease in water absorption, whereas no changes were detected in the segment perfused with supernatants obtained from the deletion mutant. Finally, partially purified toxin induced a dose dependent increment in short circuit current that reached its plateau at a toxin concentration of 4 x 10(-6) M. CONCLUSIONS: Shigella enterotoxin 1 induces a time and dose dependent intestinal secretion in the rabbit animal model, suggesting that it may be responsible for the watery phase of Shigella flexneri 2a infection.     

Enteroaggregative escherichia coli virulence factors in traveler's diarrhea strains

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhea in Spanish travelers to developing countries. In this study, the polymerase chain reaction was used to test EAEC isolates for genes encoding putative virulence factors, including EAEC adhesins, the plasmid-encoded toxin (Pet), a heat-stable enterotoxin (EAST), and Shigella enterotoxins 1 and 2 (ShET1 and ShET2). Findings included a low prevalence of genes for Pet (4.3%), ShET2 (4.3%), and the adherence factor AAF/II (8.7%). The overlapping genes encoding the ShET1 and the Pic mucinase were present in most EAEC strains tested (56.5%); however, some strains that carried this locus did not produce both proteins, as determined by Western immunoblot. Surprisingly, ShET1 and ShET2 genes were also found in other E. coli pathotypes, as was the EAST toxin locus. These findings underscore the heterogeneity of EAEC strains and suggest that the ShET1 may be an important virulence factor in traveler's diarrhea.     

福建省福氏志贺菌4c亚型的分子特征分析

目的 分析福建省福氏志贺菌4c血清亚型的毒力基因分布,菌株间的遗传相似度以及基因组的多态性。方法 运用PCR扩增技术对17株F4c菌进行ipaH、set1（set1A和set1B）、sen、ial 四种毒力基因的检测,同时运用脉冲肠凝胶电泳（PFGE）技术进行分子分型。 结果 ipaH、set1（set1A和set1B）、sen 、ial基因阳性的菌株分别占100%、94.12%、 82.35%、88.23%; 17株F4c菌分为I-IV型不同的毒力基因分布模式, I型是优势模式占76.47%;按100%的相似度,PFGE有12种型别（P1-P12）,不存在集中优势的PFGE型别;根据TENOVER原则,分为3个PFGE群(G1-G3),G1是优势群,占64.71%。结论 我省分离的F4c血清亚型的志贺菌大部分菌株具有较强的毒力特征;绝大多数感染患者之间无明显的传播关系,不同克隆群在传播中存在着集中趋势,提示某一克隆群的F4c菌有可能逐步演变成为我省主要且稳定的福氏志贺菌流行菌株。     

Deletion of the Shiga toxin gene in a chlorate-resistant derivative of Shigella dysenteriae type 1 that retains virulence

We used a probe specific for detecting the structural-gene sequences of Shiga toxin to analyze the genetic nature of toxin synthesis in mutant derivatives of Shigella dysenteriae type 1. A chlorate-resistant (chl) mutant (725-78) of S. dysenteriae type 1 strain 3818T, which had retained virulence but had lost production of high levels of cytotoxic activity associated with Shiga toxin synthesis, contained a complete deletion of the Shiga toxin structural-gene sequences. These structural-gene sequences were also absent in a derivative of S. dysenteriae type 1 that contained a substitution of Escherichia coli DNA in the trp region of the chromosome. Isolates of Shigella flexneri and Shigella sonnei also did not react with the probe. The low-level cytotoxic activities associated with the mutant S. dysenteriae type 1 strains or with the virulent S. flexneri and S. sonnei strains are neutralizable with antiserum to Shiga toxin; however, these cytotoxic activities are not determined by the genes encoding classic Shiga toxin.     

福建省2005-2010年志贺菌分离株的毒力基因分析

目的 通过检测福建省志贺菌的毒力基因,了解我省不同志贺菌菌群及血清型的毒力基因分布情况以及流行模式,评估不同菌型的危害性,为菌痢防控工作提供科学的实验依据。方法 运用PCR扩增技术,对2005-2010年福建省腹泻病人中分离的104株志贺菌进行ipaH、set1（set1A和set1B）、sen、ial 四种毒力基因的检测,分析各毒力基因的阳检率以及毒力基因的分布模式。结果 ipaH、set1（set1A和set1B）、sen 、ial毒力基因的阳检率分别为100%、36.54%、 66.35%、64.42%,其中set1基因在B群（福氏志贺菌）和D群（宋内志贺菌）的阳检率分别为85%和6.25%;104株志贺分为8种毒力基因分布模式命名为I-VIII, IV型是B群志贺菌的优势基因携带模式（67.5%）,VI型是D群志贺菌优势基因携带模式（39.06%）,同时D群中I型、VII型和V型基因模式分布也较高。结论 ipaH可作为志贺菌属的鉴定基因,set1毒力基因主要存在于福氏志贺菌（B群）中;B群志贺菌有一个集中优势的基因流行模式IV型,D群有较多的相对优势基因流行模式;说明在志贺菌进化过程中存在复杂分化形式,同时毒力基因的插入和缺失在菌型的变异和分化中有一定的相关性。     

Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus.

We have cloned the Staphylococcus aureus entB gene in Escherichia coli, using pBR322 as the vector plasmid; however, no detectable staphylococcal enterotoxin B (SEB) was produced by the E. coli clones. When the entB gene was placed downstream from the strong lambda phage promoter, PR, SEB was synthesized at readily detectable levels in E. coli. Interestingly, mature SEB was almost exclusively present in the cytoplasmic fraction. The SEB precursor was found associated with the cell membrane. The entB gene was introduced back into S. aureus, and the clones were shown to produce SEB. The entB gene has been located to a 2.1-kilobase-pair region. Maxam-Gilbert sequencing of a part of the entB gene yielded a DNA sequence that corresponds to the known amino acid sequence of SEB. Southern hybridization experiments showed that the entB gene was present on identical restriction fragments in the chromosomes of SEB-producer strains. The entB gene is absent from SEB-nonproducer strains.     

Effect of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys to subsequent infection with Shigella sonnei

All virulent shigellae have large plasmids. Plasmid-associated genes encode the expression of membrane-associated proteins (MAP), some of which correlate with the ability to invade susceptible epithelial cells. These MAP are serologically related in all of the shigella serotypes and evoke an antibody response after infection. To determine whether the MAP have a significant role in protection, 24 monkeys were infected with virulent Shigella flexneri 2a. After recovery, one group (with controls) was rechallenged with S. flexneri 2a; another group (with controls) was fed Shigella sonnei. The animals that were rechallenged with S. flexneri 2a were protected, while those that were fed S. sonnei experienced the same incidence of disease as controls. No differences in serum immune response to MAP after primary infection with S. flexneri were detected in immunoblots using lysates of S. flexneri or S. sonnei or in ELISA using water extracts of these strains.