基于质量源于设计理念在伤寒沙门菌中试规模培养工艺放大中的应用

目的基于质量源于设计(quality by design, QbD)理念,确定伤寒沙门菌(Salmonella typhi)中试规模(200 L)的培养工艺。方法运用QbD通过分析伤寒沙门菌培养工艺核心指标、培养过程,以确定对核心指标可能产生影响的因素。采用单因素试验对pH控制值、溶氧控制值和补料方式等进行考察,通过中试规模(20 L)培养工艺桥接和中试规模(200 L)培养工艺的放大,分析中试规模培养条件及发酵液中伤寒Vi荚膜多糖含量等。结果连续培养了6批次200 L规模发酵液,培养8 h时发酵液中伤寒Vi荚膜多糖含量均≥43.33μg/mL(期望值);培养条件:(1) pH控制值为7.2;(2)溶氧控制值为35%;(3)补料方式为培养初始补加葡萄糖溶液使其质量浓度为3 g/L,培养过程中补加葡萄糖溶液使其质量浓度保持在1～3 g/L。结论通过分析200 L规模培养过程监测数据,成功在中试规模(200 L)完成伤寒沙门菌培养工艺放大。     

沙门氏伤寒菌荚膜多糖抗伤寒的研究-尼泊尔现场报告

<正> 伤寒在发展中国家是一种多发的烈性病。过去通过使用清洁水、污水处理和对患者及未发病的病菌携带者进行及时诊断、隔离和治疗等方法控制这种病的流行,但今后几十年内,局部或大面积流行此病的国家仅用这些方法还不够。对发展中国家来说,伤寒是最常见的烈性传染病。从病原学角度看,沙门氏菌属中唯有沙门氏伤寒菌具有荚膜多糖(Vi抗原),而沙门氏副伤寒菌C株偶尔才有同样的荚膜抗原。     

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。     

一种防止肺炎链球菌和伤寒沙门菌致病的双价疫苗

<正>在发展中国家的儿童中,肺炎球菌和伤寒沙门菌感染是两种主要疾病,对于伤寒,已上市的Vi多糖疫苗在2岁以下的儿童中是无效的,虽然据调查Vi结合疫苗已经在临床试验中是有效的,但它们目前只适用于少数地区。肺炎球菌荚膜多糖结合疫苗对儿童是非常有效的,但受到一些限制,包括成本高和有限的血清型覆盖率,我们先前已经表明,由肺炎链球菌融合蛋白PsaA和与多糖结合的肺炎链球菌     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的:鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法:用LB(Luria-Bertani,LB)培养基和TSB(Tryptose Soya Broth,TSB)培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48 h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜(Confocal Laser scanning microscopy,CLSM)观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果:用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论:鼠伤寒沙门菌pStSR100质粒能促进该菌在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

鼠伤寒沙门菌感染巨噬细胞铁稳态相关基因mRNA表达谱

用荧光定量PCR法检测鼠RAW264.7巨噬细胞感染与未感染鼠伤寒沙门菌后18种铁穗态相关基因的表达,评估宿主与病原体相互作用中铁稳态效应。研究显示,活的鼠伤寒沙门菌感染巨噬细胞1 h后可以诱导转铁蛋白受体表达,引起细胞内动态铁池相关基因的mRNA水平上长。基因表达分析显示,沙门菌通过诱导铁氧还原酶(Steap3)、铁膜转运蛋白(Dmt1)、铁调节因子Tfr2/Hfe以及铁调节蛋白(Irp1和Irp2)的表达主动吸收铁,而经铁转运蛋白(Fpn1)的铁外流并无明显改变。沙门菌在感染后1h积极地驱动了转铁蛋白介导的铁吸收程序。     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的：鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法：用LB（Lufia—Bertani,LB）培养基和TSB（TryptoseSoyaBroth,TSB）培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜（ConfocalLaserscanningmicroscopy,CLSM）观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果：用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论：鼠伤寒沙门菌pStSR100质粒能促进该茵在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

鼠伤寒沙门菌六型分泌系统效应蛋白Clpv对巨噬细胞极化的影响北大核心

【目的】探讨Ⅵ型分泌系统(typeⅥsecretion system,T6SS)效应蛋白Clpv在鼠伤寒沙门菌(Salmonella enterica serovar Typhimurium)致病过程中的功能。【方法】以鼠伤寒沙门菌SL1344基因组为模板克隆clpv基因,并比较与其他革兰氏阴性菌台湾假单胞菌(Pseudomonas taiwanensis)、植生拉乌尔菌(Raoultella planticola)、鳗利斯顿氏菌(Listonella anguillarum)、菠萝多源菌(Pantoea ananatis)、粘放线菌(Actinomyces viscosus)和大肠埃希菌(Escherichia coli)的同源性;将clpv基因克隆至pEGFP-N1载体构建重组质粒pEGFP-Clpv,利用Western blotting、实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,q-PCR)、荧光显微镜以及流式细胞术检测蛋白表达、定位及诱导小鼠巨噬细胞M1型和M2型极化水平。【结果】clpv基因全长为2637 bp,与台湾假单胞菌的同源性最高;Western blotting、qPCR和免疫荧光检测表明重组蛋白大小约120 kDa,在细胞中有明显绿色荧光并且主要定位于细胞膜;q-PCR和流式细胞术结果发现Clpv转染组巨噬细胞M1型极化显著增加(P<0.01),M2型巨噬细胞极化显著减少(P<0.01)。【结论】成功克隆表达鼠伤寒沙门菌T6SS效应蛋白Clpv,并明确其胞内表达定位以及对巨噬细胞极化的影响。     

6种沙门菌单克隆抗体的制备和效能评价

目的：制备稳定、特异、高亲和性的分别针对甲型副伤寒沙门菌、乙型副伤寒沙门菌、丙型副伤寒沙门菌、肠炎沙门菌、伤寒沙门菌和猪霍乱沙门菌的单克隆抗体。方法：用甲醛灭活的菌液抗原免疫BALB／c小鼠,取脾细胞与SP2／0骨髓瘤细胞融合;用灭活的菌液包被酶标板,ELISA筛选阳性克隆株,建立细胞系;选取高效分泌杂交瘤细胞,常规制备腹水并纯化,进行单抗特异性与亲和性评价。结果：筛选得到分泌6种沙门菌相应单克隆抗体的杂交瘤细胞株,获得高亲和性单抗;所有单抗与大部分病原菌（包括7种沙门菌、3株志贺菌、2株李斯特菌、4株致病性大肠杆菌、2株霍乱弧菌）无交叉反应,但由于同类型O抗原的广泛分布,抗乙型副伤寒沙门菌单抗与鼠伤寒沙门菌、抗伤寒沙门菌单抗与肠炎沙门菌有明显的交叉反应。结论：沙门菌单抗的制备,为感染性腹泻的监测、诊断奠定了基础。     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

Vi antigen of <Emphasis Type="Italic">Salmonella</Emphasis> enetrica serovar Typhi — biosynthesis,regulation and its use as vaccine candidate

Vi capsular polysaccharide (Vi antigen) was first identified as the virulence antigen of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever in humans. The presence of Vi antigen differentiates S. Typhi from other serovars of Salmonella. Vi antigen is a linear polymer consisting of α-1,4-linked-N-acetyl-galactosaminuronate, whose expression is controlled by three chromosomal loci, namely viaA, viaB and ompB. Both viaA and viaB region are present on Salmonella Pathogenicity Island-7, a large, mosaic, genetic island. The viaA region encodes a positive regulator and the viaB locus is composed of 11 genes designated tviA-tviE (for Vi biosyhthesis), vexA-vexE (for Vi antigen export) and ORF 11. Vi polysaccharide is synthesized from UDP-N-acetyl glucosamine in a series of steps requiring TviB, TviC, and TviE, and regulation of Vi polysaccharide synthesis is controlled by two regulatory systems, rscB-rscC (viaA locus) and ompR-envZ (ompB locus), which respond to changes in osmolarity. This antigen is highly immunogenic and has been used for the formulation of one of the currently available vaccines against typhoid. Despite advancement in the area of vaccinology, its pace of progress needs to be accelerated and effective control programmes will be needed for proper disease management.     

Molecular Characterization of the viaB Locus Encoding the Biosynthetic Machinery for Vi Capsule Formation in Salmonella Typhi

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi.     

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.     

Detection of O-acetylated Vi polysaccharide of Salmonella enterica subspecies typhi by enzyme immunoassay.

Immunisation with capsular Vi polysaccharide (Vi PS) of Salmonella enterica serovar Typhi (S. typhi) protects against typhoid. This protection depends on the presence of O-acetyl groups on the Vi PS, which form an immunodominant epitope. An antiserum raised against conjugated Vi PS was used as the basis for an indirect Enzyme Immunoassay (EIA). The antiserum did not react with lipopolysaccharide of five gram negative bacteria including S. typhi. Vi PS from three different sources was tested, and all but one of 18 native Vi PS preparations had EIA values comparable to a standard Vi PS preparation. The sensitivity of the EIA for the detection of O-acetyl groups on Vi PS was compared to an NMR spectroscopy assay (Biologicals 28 (2000) 17-24). The EIA distinguished between O-acetylated and de-O-acetylated Vi PS preparations. However, significantly lower EIA reactivity was observed only for samples which had O-acetylation levels of 25% or less. This assay should facilitate batch control of Vi vaccines.     

Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi.

Plasmid pGBM124, which contains a 14-kb Salmonella typhi chromosomal DNA fragment capable of producing the Vi antigen in Escherichia coli HB101 and ViaB-deleted S. typhi GIFU 10007-3, was studied. We determined the complete nucleotide sequence of this fragment and found 11 open reading frames. Mutagenesis, subcloning, and complementation analysis showed that three genes (vipA, vipB, and vipC) are involved in biosynthesis of the Vi polysaccharide. The putative primary amino acid sequence suggests that both vipA and vipB encode the NAD- or NADP-dependent enzymes to synthesize the nucleotide sugar for the Vi polysaccharide. Five genes (vexA, vexB, vexC, vexD, and vexE) may be involved in translocation of the Vi polysaccharide. Proteins VexA, VexB, VexC, and VexD had moderate similarities to components of group II capsule transporters, and the VexC protein had a putative ATP-binding site. These data indicate that the transport system for the Vi polysaccharide belongs to the ATP-binding cassette transporters. By using the isogenic Vi+ and Vi- strains constructed in this study, we reconfirmed that the Vi antigen is necessary for the serum resistance of S. typhi.     

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.