Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes

Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 10(5) cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.     

Nonharsh methods of isolation of antigenic complexes of Klebsiella pneumoniae, and analysis of their protective activity

Protective antigenic complexes capable of protecting mice from septic Klebsiella infection can be obtained from Klebsiella pneumoniae. The preparations obtained by the method of aqueous extraction and by the action of hydroxyl amine and autolysis have been found to possess approximately the same protective activity and to protect mice from infection with Klebsiella pneumoniae strains belonging to both homologous and heterologous serotypes.     

Effect of a hydroxylamine Klebsiella vaccine on the rate of elimination of the causative agent and on indices of humoral immunity

Immunization with hydroxylamine Klebsiella vaccine ensures the elimination of K. pneumoniae from the blood, the lungs, the liver, the kidneys and the spleen of mice and induces considerable immunological shifts, manifested by a rise in the level of specific antibodies and by an increase in the preventive potency of the blood serum.     

Protective activity of a cell-free Klebsiella vaccine in relation to different Klebsiella pneumoniae serovars

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial cells with hydroxylamine, for protecting mice from Klebsiella septic infection caused by the homologous serovar and 9 heterologous serovars of K. pneumoniae was studied. The newly developed preparation was found capable of stimulating immunity not only to the homologous K. pneumoniae serovar, but also to other K. pneumoniae heterologous serovars: K1, K9, K11, K16, K20, K61. The protective capacity of the preparation with respect to these serovars was not inferior to that of the vaccines prepared by the same method from the corresponding homologous strains. The capacity of the vaccine to protect mice from Klebsiella sepsis was manifested irrespective of the virulence of the strains used for challenge.     

肺炎克雷伯杆菌疫苗中试生产中细菌培养工艺的改进及优化

目的中试生产中对肺炎克雷伯杆菌培养工艺进行改进及优化。方法采用液体综合培养基代替半综合培养基在10L和100L中国丽生物反应器中对肺炎克雷伯杆菌进行培养,在10L中国丽生物反应器探讨不同的培养基配方、pH值、培养温度、搅拌转速、溶氧,工艺参数稳定后,扩大培养到100L中国丽生物反应器,并探讨培养过程中补加葡萄糖的浓度及补加方式等对细菌浓度及荚膜多糖含量的影响。结果肺炎克雷伯杆菌液体综合培养基可代替半综合培养基用于该菌的培养,培养过程中维持pH值7．2、温度37℃、通气60L／h、搅拌转速250r／min、培养到2h时开始以恒速补加30mL／L40％葡萄糖溶液、培养时间为5h,细菌长势最好,收获的荚膜多糖含量最高。结论肺炎克雷伯杆菌的培养工艺放大到100L中国丽生物反应器中,经过多次试验初步建立了稳定的肺炎克雷伯杆菌中试培养工艺。     

Experimental study of the immunogenicity of a monopreparation of Klebsiella pneumoniae antigen complex and its association with Staphylococcus, Proteus and Escherichia coli antigens

The study carried out in mice with experimental Klebsiella sepsis has revealed that Staphylococcus, Proteus and E. coli antigenic complexes used as monovaccines ensure the protection of a definite percentage of the animals from K. pneumoniae infection. The mixture of these 3 preparations possesses a higher protective potency. The immunogenic potency of K. pneumoniae antigenic complex used as a component of combined vaccines with 2 or 4 components has proved to be sufficiently high and not inferior to the potency of K. pneumoniae monovaccine.     

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.     

Study of the mode of action of ribosomal vaccines from Klebsiella and Streptococcus pneumoniae and their ribonucleic and protein fractions using passive immunization

The vaccinating potency of ribosomal fractions of Klebsiella pneumoniae and Streptococcus pneumoniae as well as their ribosomal RNA and protein fractions has been studied with respect to their ability to induce cellular or humoral immunity. Experiments with transfer of serum or spleen cells from vaccinated animals have shown that anti-Klebsiella immunity is essentially cellular, while streptococcal immunity is exclusively humoral. Results have been discussed as a function of differential results for the various fractions under study.     

Pneumococcal surface protein A and new approaches for pneumococcal vaccine development

The problem of pneumococcal infections is pressing for the whole world. Existing vaccines based only on pneumococci polysaccharide antigens or polysaccharide antigens and diphtherial anatoxin are not capable of protecting from all serotypes of the microorganism. Reasonability of creation of pneumococcal vaccine based on surface proteins of Streptococcus pneumoniae is discussed in the literature. One of such key pneumococcal proteins is pneumococcal surface protein A (PSPA), because it is detected in all the S. pneumoniae strains, has cross activity and switches B-cell immune response to T-cell. Currently the development of conjugated vaccine based on surface proteins and capsule polysaccharides of pneumococcus seems promising.     

PCR鉴定肺炎克雷伯菌的强毒性血清型

目的:了解肺炎克雷伯菌强毒性血清型K1、K2、K54和K57型菌株在我国重庆、北京、深圳三地的分布及流行趋势。方法:采用PCR对310株肺炎克雷伯菌临床分离株进行血清型K1、K2、K54和K57检测。结果:310株菌中,K1、K2、K54和K57血清型分别占14.2%、9.4%、6.5%和4.2%;来自呼吸系统标本分离株中的K1血清型菌株在4种检测的强毒血清型中占首位,为呼吸系统总数的17.4%。结论:310株肺炎克雷伯菌的4种强毒性血清型中,K1血清型菌株所占比例高,较为流行。     

乳及乳制品中肺炎克雷伯氏菌PCR-DHPLC检测新技术的建立

为了应用PCR结合变性高效液相色谱(DHPLC)技术建立乳品中肺炎克雷伯氏菌的快速检测方法,根据肺炎克雷伯氏菌16S-23S rRNA特异基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以肺炎克雷伯氏菌等57株参考菌株做特异性试验;将肺炎克雷伯氏菌菌株稀释成不同梯度,做灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到100 CFU/mL,可以快速、准确检测肺炎克雷伯氏菌,是乳及乳制品中致病菌快速检测的新技术。     

血流感染产ESBLs肺炎克雷伯菌毒力基因及 分子分型研究

目的研究血流感染产ESBLs肺炎克雷伯菌的毒力基因和基因分型特点。方法采用PCR检测菌株中高毒力因子、荚膜血清型以及ST分型;采用微量肉汤稀释法对菌株进行药敏试验;采用加克拉维酸的复合药(头孢他啶/克拉维酸或头孢噻肟/克拉维酸)与单药(头孢噻肟或头孢他啶)的药敏纸片组合进行肺炎克雷伯菌产ESBLs的表型确证试验。结果 128株血流感染肺炎克雷伯菌中,有23株产ESBLs(产ESBLs组),占17.97%(23/128);105株不产ESBLs(非产ESBLs组),占82.03%(105/125)。本地区血流感染肺炎克雷伯菌主要流行ST型别为ST23、ST65、ST37和ST29,其中ST23、ST29、ST65为非产ESBLs的优势ST型别菌株,而在产ESBLs菌株中无优势型别。两组菌在高黏液表型、荚膜血清型和毒力基因分布上差异均无统计学意义(P0.05)。产EBSLs组中发现8株高毒力产EBSLs肺炎克雷伯菌。结论临床诊疗中需在肺炎克雷伯菌耐药株中识别出高毒力肺炎克雷伯菌并给与及时的治疗,避免其并发症的发生。     

Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.     

肺炎链球菌假想蛋白SPD0873的表达纯化及保守性分析

目的通过构建原核表达载体,获得纯化的肺炎链球菌S．pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S．pn菌株中的保守性。方法分离培养D39型肺炎链球菌,获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32（a）内,测序鉴定。将重组质粒转化到E．coli Rossetta（DE3）中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni—NTA树脂纯化后,获得的重组蛋白用SDS—PAGE和Western印迹鉴定;将鉴定后纯化的蛋白免疫BALB／C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达。纯化蛋白免疫BALB／C小鼠获得高滴度、高特异性的的多克隆抗体,Western印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达。结论成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。     

Antigenic structure of the polysaccharide capsule of Streptococcus pneumoniae strains isolated in Leningrad 1978-1984

The serotyping of 826 S. pneumoniae strains, isolated in conditionally diagnostic concentrations from the bronchial contents of patients with acute and chronic inflammatory pulmonary diseases during 1978-1984 in Leningrad, was made. The study revealed the prevalence of serotypes and groups 6, 23, 9, 3, 19, 15 and the undulant character of fluctuations in their annual occurrence. The specific proportion of the prevailing serotypes of S. pneumoniae among the cultures isolated from patients with acute pneumonia and acute bronchitis (6 and 19) was found to differ from that among S. pneumoniae strains isolated from patients with chronic bronchitis; in the latter patients serotypes 3 and 9 occurred more frequently (P less than 0.01).     

Construction of a multivalent vaccine strain of Shigella flexneri and evaluation of serotype-specific immunity

Shigella flexneri causes more fatalities by shigellosis than any other Shigella species. There are 13 different serotypes of S. flexneri and their distribution varies between endemic geographical regions. The immune response against S. flexneri is serotype-specific, so current immunization strategies have required the administration of multiple vaccine strains to provide protection against multiple serotypes. In this study, we report the construction of a multivalent S. flexneri vaccine strain, SFL1425, expressing the O-antigen structure specific for serotypes 2a and 5a. This combination of type antigens has not previously been reported for S. flexneri. The multivalent vaccine strain, SFL1425 was able to induce a specific immune response against both serotypes 2a and 5a in a mouse pulmonary model.     

Protective efficacy of combined administration of the multicomponent vaccine and the immunomodulator myelopid in experimental infections in mice

The influence of myelopid (MP) on the protective activity of polycomponent vaccine VP-4 prepared from the antigens of opportunistic bacteria was studied on experimental infections of mice, caused by Klebsiella pneumoniae, Salmonella typhimurium and Staphylococcus aureus. In staphylococcal and Klebsiella infections the joint administration of vaccine VP-4 and MP produced more pronounced protective effect than each of these preparations, introduced alone. The protective action of vaccine VP-4 was specially enforced by MP in cases of local staphylococcal infection. Recommendations on the joint use of two or more immunomodulating agents are possible only on the basis of the experimental substantiation of their effect in definite infections.     

Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

Potential Pathogens in the Environment: Klebsiella pneumoniae, a Taxonomic and Ecological Enigma

A nitrogen-deficient medium and m-Endo agar were employed in the isolation of members of the tribe Klebsielleae from surfaces of vegetables and seeds. With m-Endo agar at an incubation temperature of 37 C, nearly 50% of the vegetables and seven out of seven seed samples yielded organisms which biochemically and serologically were identified as Klebsiella pneumoniae, Viable counts were generally in the range of 10(3) cells per g of vegetable peel or seed. Organisms classified as K. pneumoniae exhibited seven different IMViC patterns, with the --++, ++++, and -+++ patterns most common. Seven of the eleven K. pneumoniae serotypes encountered have previously been isolated from human urinary tract and other infections. Fifty percent of the 40 K. pneumoniae examined exhibited positive acetylene-reducing activity, i.e., they possessed the capability for fixing N(2). Vegetables containing K. pneumoniae may constitute a potential reservoir for human nosocomial genitourinary or other infections.