Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

伤寒沙门菌动物模型研究进展

目前伤寒沙门菌引起的人类伤寒仍是一种严重危害人类健康的疾病,且近年来多重耐药伤寒沙门菌株频频出现,使伤寒的治疗更加棘手.由于该菌具有严格的宿主特异性,又缺乏理想的动物模型,其致病机制的研究、疫苗及药物的研发受制约.新近研究发现,免疫系统人源化小鼠模型和诱导型一氧化氮合酶基因敲除小鼠模型可用于伤寒沙门菌的体内实验.本文就其应用现状及缺憾作一综述.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

Reduced amounts of LPS affect both stress tolerance and virulence of Salmonella enterica serovar Dublin

Signature-tagged mutagenesis (STM) is a widely used technique for identification of virulence genes in bacterial pathogens. While this approach often generates a large number of mutants with a potential reduction in virulence a major task is subsequently to determine the mechanism by which the mutations influence virulence. Presently, we have characterised a Salmonella enterica serovar Dublin STM mutant that, in addition to having reduced virulence, was also impaired when growing under various stress conditions. The mutation mapped to the manC (rfbM) gene of the O-antigen gene cluster involved in O-antigen synthesis. The O-antigen is a component of the lipopolysaccharide (LPS) forming a unique constituent of the outer membrane of Gram-negative bacteria. While mutations in the O-antigen genes usually eliminate the entire O-antigen side chain we found that the transposon mutant produced intact O-antigen, however, the mutation reduced the amount of LPS.     

Competition among isolates of Salmonella enterica ssp. enterica serovar Typhimurium: role of prophage/phage in archived cultures

Previously, we reported extensive diversity among survivors of Salmonella enterica ssp. enterica serovar Typhimurium that were stored for four decades in sealed agar stabs. Thus raising the question: was there selection for greater fitness among eventual survivors? To address this, we cocultured archived LT2 survivors with nonarchived (parental) LT2 strains in competition experiments. Selected archived strains outgrew a nonarchived LT2 sequenced strain. Although we initially assumed this was the result of mutations empowering greater nutritional utilization, we found phage selection was also involved. Phage fels- 1 and fels- 2 in supernatants were identified by primer/PCR as a putative selective force following single plaque isolations on a prophage-free strain and testing on appropriate hosts. In confirmatory experiments, instead of coculture in Luria–Bertani requiring antibiotic marker insertions, competing strains without markers were inoculated at opposite edges of motility plates. Not only did the archived LT2 population overgrow the nonarchived LT2 population, but also clear zones appeared at edges of encounters from which phage fels- 1 and fels- 2 (but not gifsy- 1 nor gifsy- 2) were recovered. However, in competitions of an archived strain with S . Typhimurium ATCC 14028, phage emerged that had a DNA base sequence segment of prophage ST64B but the sequence differed from the reported homologous segment in ST64B.     

肠道菌群代谢产物在鼠伤寒沙门菌感染中的作用研究进展

人和动物肠道内生存着多种多样的微生物群体,它们与宿主共同进化,对宿主的健康至关重要。肠道菌群可以发酵宿主难以消化的复杂碳水化合物,为宿主肠道细胞提供能量,同时其代谢产物对肠道病原菌沙门菌的感染产生着重要影响。正常情况下,肠道菌群代谢产物如丁酸与丙酸可以抑制沙门菌在肠道中的定植或者毒力基因的表达,而在肠道菌群受到扰乱时,其代谢的琥珀酸盐和1,2-丙二醇等物质却能促进沙门菌增殖。近年来,越来越多的研究揭示了肠道菌群代谢产物对沙门菌感染的影响。本综述通过总结近年来关于鼠伤寒沙门菌入侵时肠道菌群代谢产物改变的研究,综合阐述了肠道菌群代谢产物影响沙门菌感染的机制。     

重组SEF21脂质体口服疫苗对肠炎沙门菌感染的作用

目的探讨制备脂质体包裹重组SEF21疫苗,并评价其在预防肠炎沙门菌(S.enteritidis)感染中的作用。方法利用PCR获得SEF21基因,并连接至pET-28a(+)载体。将pET-28a(+)-SEF21在BL21(DE3)大肠埃希菌中表达,通过镍层析柱纯化高表达的rSEF21蛋白。制备脂质体包裹rSEF21疫苗,并对鸡进行2次免疫,然后利用S.enteritidis进行攻毒实验。ELISA检测血清以及肠内容物中的抗体效价。结果所有被免疫鸡的血清及肠黏液中产生了高效价的IgG和IgA抗体。脂质体包裹rSEF21所免疫的鸡的粪便样本中S.enteritidis数量明显下降。结论口服脂质体包裹的重组SEF21蛋白疫苗能有效保护鸡对抗S.enteritidis感染。     

Effects of yeast probiotic formulation on viability, revival and protection against infection with Salmonella enterica ssp. enterica serovar Typhimurium in mice

Aims:  To compare the effects of five yeast probiotic formulations on viability, revival and washout kinetic in the digestive tract of mice, and the protection against an experimental infection with Salmonella enterica serovar Typhimurium.
Methods and Results:  The number of viable cells in five commercial probiotic products codified as A, B, C and D ( Saccharomyces boulardii – lyophilized) and E ( Saccharomyces cerevisiae – aqueous suspension) was determined, as well as revival and washout kinetic in mouse intestine. Protective capacity was evaluated by survival rate and histopathology of liver and intestine of mice treated with each product and then challenged with Salm . Typhimurium.
Conclusions:  Product A contained the highest number of viable cells and, fed to mice, gave the highest counts of viable yeasts and the longest persistence in faeces. Probably as a consequence, the highest survival and protection of intestinal and hepatic tissues were observed when product A was used for mouse treatment. Product E showed low counts in the formulation and was not recovered from mouse intestine.
Significance and Impact of the Study:  Formulation (lyophilization or aqueous suspension) is an important factor for revival and survival of a probiotic product in vivo and consequently for its protective properties.     

Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross‐protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild‐type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild‐type strain were 3.0 × 10⁸ CFU and 1.9 × 10³ CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild‐type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti‐LPS and anti‐flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild‐type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.     

伤寒沙门菌非编码RNA617的分子鉴定及其对生物膜形成的影响研究

本研究旨在探讨伤寒沙门菌(Salmonella enterica serovar Typhi, S. Typhi)中非编码RNA617(non-coding RNA617,ncRNA617)的分子特性,并研究其对生物膜形成的影响及作用机制。采用Northern blot方法检测ncRNA617的表达,通过cDNA 5’末端快速扩增技术(5’-rapid amplification of cDNA end,5’RACE)和逆转录-聚合酶链式反应(reverse transcriotion-polymerase chain reaction,3’RT-PCR)实验分析ncRNA617可能的转录起始位点和终止位点;构建ncRNA617缺陷菌株、回补菌株和过表达菌株等相关菌株,通过生物膜形成实验,观察ncRNA617对伤寒沙门菌生物膜形成的影响,并用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)分析生物膜形成相关基因表达水平的变化,综合运用生物信息学方法预测ncRNA617和差异基因的结合区域,初步分析ncRNA617发挥调控作用的机制。结果显示,伤寒沙门菌确有ncRNA617的表达,长度约300 nt,其转录起始位点位于mig-14终止密码子下游967 nt处,终止位点位于t2681起始密码子上游 2 378～2 560 nt处。与野生对照菌株相比,ncRNA617缺陷菌株生物膜形成能力增强(P<0.05),回补菌株的生物膜形成能力恢复至野生菌株水平,过表达菌株的生物膜形成能力有所下降(P<0.05)。qPCR结果表明,ncRNA617可负向调控多个生物膜形成相关基因的转录表达水平(P<0.05)。经生物信息学方法预测发现,ncRNA617与差异基因有不同的结合区域。本研究结果提示,ncRNA617在伤寒沙门菌中存在,其长度约270～452 nt。ncRNA617可能通过靶向结合生物膜形成相关基因下调基因表达,从而负向调控伤寒沙门菌生物膜的生成。     

Epithelial invasion by Salmonella Typhi using STIV–Met interaction

Typhoid is a life‐threatening febrile illness that affects ~24.2 million people worldwide and is caused by the intracellular bacteria Salmonella Typhi (S. Typhi). Intestinal epithelial invasion by S. Typhi is essential for the establishment of successful infection and is traditionally believed to depend on Salmonella pathogenicity island 1‐encoded type 3 secretion system 1 (T3SS‐1). We had previously reported that bacterial outer membrane protein T2942/STIV functions as a standalone invasin and contributes to the pathogenesis of S. Typhi by promoting epithelial invasion independent of T3SS‐1 (Cell Microbiol, 2015). Here, we show that STIV, by using its 20‐amino‐acid extracellular loop, interacts with receptor tyrosine kinase, Met, of host intestinal epithelial cells. This interaction leads to Met phosphorylation and activation of a downstream signalling cascade, involving Src, phosphatidylinositol 3‐kinase/Akt, and Rac1, which culminates into localized actin polymerisation and bacterial engulfment by the cell. Inhibition of Met tyrosine kinase activity severely limited intestinal invasion and systemic infection by S. Typhi in vivo, highlighting the importance of this invasion pathway in disease progression. This is the first report elucidating the mechanism of T3SS‐1‐independent epithelial invasion of S. Typhi, and this crucial host–pathogen interaction may be targeted therapeutically to restrict pathogenesis.     

Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.     

利用间接ELISA定量分析伤寒沙门菌表面呈现表达的流感病毒抗原

目的:建立定量检测伤寒沙门菌表面呈现表达的流感病毒抗原的间接ELISA方法。方法:ELISA板以2.5%的戊二醛溶液预处理,将呈现表达M2e等流感病毒抗原表位的伤寒沙门菌的全细胞抗原在ELISA板上进行干燥包被,通过间接ELISA确立全菌抗原的最佳包被浓度;分别采用化学合成多肽M2e和GST-M2e融合蛋白干燥包被ELISA板,绘制标准曲线,对沙门菌表面呈现表达的抗原进行定量分析;对多肽和融合蛋白干燥包被进行比较,同时确立用于定量表面展示量的回归方程。结果:用多肽包被测定的呈现表达的M2e分子数为9.8×104,以GST-M2e包被测定的呈现表达的M2e分子数为1.3×105。结论:利用全菌干燥包被ELISA板可以对伤寒沙门菌表面呈现表达的抗原进行很好的定量分析。     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

高盐环境下鼠伤寒沙门氏菌差异糖蛋白富集及组学分析

【背景】沙门氏菌是一种革兰阴性肠道病原菌,主要依靠III型分泌系统(typeIIIsecretion systems,T3SSs)来产生与致病性相关的效应蛋白。其中沙门氏菌致病岛(Salmonellapathogenicity island,SPI)区域是关键的基因区域。高盐浓度条件可以诱导SPI-1上效应蛋白的表达。【目的】探究在高盐浓度条件下鼠伤寒沙门氏菌(SalmonellaentericaserovarTyphimurium)糖蛋白的差异表达情况,寻找有意义的效应糖蛋白。【方法】将鼠伤寒沙门氏菌在普通培养基和高盐培养基中培养,收集菌体并超声裂解,提取蛋白后,用肼偶联法富集糖蛋白并用胰酶酶解,通过二甲基标记定量及LC/MS定量蛋白质组学方法进行糖蛋白的定量,用Thermo Proteome Discoverer 2.2软件对标记蛋白进行定性及定量分析。【结果】质谱结果显示,高盐环境中,沙门氏菌有19个糖蛋白的表达发生显著改变,其中,上调蛋白10个,最为显著的是ompC基因编码的外膜孔蛋白;下调表达糖蛋白9个,最为显著的是yjgF基因编码的翻译起始抑制因子。【结论】根据定量蛋白质组...     

Septin‐regulated actin dynamics promote Salmonella invasion of host cells

Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.