Rapid PCR test for Streptococcus suis serotype 7

Recent epidemiological studies on Streptococcus suis infections in pigs indicated that, besides serotypes 1, 2 and 9, serotype 7 is also frequently associated with diseased animals. For the latter serotype, however, no rapid and sensitive diagnostic methods are available. This hampers prevention and control programs. Here, we describe the development of a type-specific PCR test for the rapid and sensitive detection of S. suis serotype 7. The test is based on DNA sequences of capsular (cps) genes specific for serotype 7. These sequences were identified by cross-hybridization of several individual cps genes with the chromosomal DNAs of 35 different S. suis serotypes.     

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

Proteome analysis of Streptococcus suis serotype 2

Outbreaks in humans, caused by Streptococcus suis serotype 2 (SS2), were reported in 1998 and 2005 in China. However, the mechanism of SS2-associated infection remains unclear. For the first time, a 2-D gel approach combined with MS was used to establish a comprehensive 2-D reference map for aiding our understanding of the pathogenicity of SS2. The identification of 694 out of 834 processed spots revealed 373 proteins. Most of the identified proteins were located in the cytoplasm and were involved in energy metabolism, protein synthesis, and cellular processes. Proteins that were abundant in the 2-DE gels could be linked mainly to housekeeping functions in carbohydrate metabolism, protein quality control and translation. 2-DE of secretory proteins was performed using IPG strips of pH 4-7. Among the 102 protein spots processed, 87 spots representing 77 proteins were successfully identified. Some virulence-associated proteins of SS2 were found, including arginine deiminase, ornithine carbamoyl-transferase, carbamate kinase, muramidase-released protein precursor, extracellular factor, and suilysin. Enolase and endopeptidase have been proposed as putative virulence-associated factors in this study. The 2-D reference map might provide a powerful tool for analyzing the virulence factor and the regulatory network involved in the pathogenicity of this microorganism.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

Efficient subtyping of Yersinia enterocolitica bioserotype 4/O:3 with pulsed-field gel electrophoresis

The purpose of this study was to evaluate the efficiency of pulsed-field gel electrophoresis (PFGE) for differentiation of isolates of Yersinia enterocolitica bioserotype 4/O:3 recovered from pig tongues at retail level in the Helsinki area during October and November of 1996. NotI generated 15 different PFGE patterns for 128 isolates. The discriminatory index did not exceed 74% due to the presence of two predominant PFGE-types, NA1 (58/128) and NB1 (25/128). After preliminary investigations with 35 enzymes, ApaI, XbaI, XhoI and SpeI were chosen for further characterization of the isolates. The discriminatory index increased from 74% to 93% and the number of different genotypes from 15 to 30 when isolates with the same PFGE pattern with NotI were further characterized with ApaI and XhoI, indicating that PFGE can be an efficient technique for characterization of bioserotype 4/O:3.     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 α-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest that human and animal isolates of S. dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.     

Optimization of pulse-field gel electrophoresis for Bartonella subtyping

Bartonella is a significant human pathogen and is the world's most common bacterial zoonosis acquired from companion animals. However, there is no uniform method for Pulse-Field Gel Electrophoresis (PFGE) for Bartonella population genetics studies. Further, some genes of Bartonella can mutate frequently and may affect the use of PFGE for Bartonella. Here we designed methods to solve these problems. We standardized the bacterial concentration, selected the appropriate digestion enzyme, optimized the electrophoretic parameters and characterized reproducibly two Bartonella species strains. Thus we optimized the PFGE procedure and determined how often Bartonella mutated. Our data shows a practical protocol for inter- and intra-species identification of Bartonella and was reproducible using two species strains that showed no mutation occurred after two passages for B. elizabethae; but mutation did occur in B. henselae.     

Identification of immunogenic cell wall-associated proteins of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. An immunoproteome-based approach was developed to identify candidate antigens of SS2 for vaccine development. Hyperimmune sera, convalescent sera, and control sera were analyzed for reactivity by Western Blot against SS2 cell wall-associated proteins (WAPs) separated by 2-DE. A total of 34 proteins were identified by immunoproteomic analysis, of which 15 were recognized by both hyperimmune sera and convalescent sera, including most WAPs currently characterized as SS2 vaccine candidate antigens: muramidase-released protein (MRP), surface protein SP1 (Sao), and glyceraldehyde-3-phosphate dehydrogenase (GapdH). The novel immunogenic proteins may be developed as alternative antigens for further study of SS2 vaccine and diagnostics.     

高致病性2型猪链球菌毒素-抗毒素系统SezAT

<正>高致病性2型猪链球菌(Streptococcus suis serotype 2,SS2)属于革兰氏阳性B组链球菌,是一种重要的人畜共患传染病病原菌,它不仅可以导致猪出现急性败血症、脑膜炎、关节炎、心内膜炎及急性死亡,还可以通过伤口和呼吸道等传播途径,导致人的感染。自1998及2005年我国发生两次2型猪链球菌大流行后,该菌所引起的链球菌毒素休克综合征引起国际高度重视,我国不少学者聚焦该领域的研究[1]。本刊2012年第2期刊登了王敏、胡福泉等的文章"高致病性2型猪链球菌毒-抗素毒素系统SezAT的鉴     

2型猪链球菌新毒力因子PlcR的发现

<正>2型猪链球菌(SS2)是一种重要的人畜共患传染病病原体。SS2感染不仅可致猪急性败血症、脑膜炎、关节炎、心内膜炎及急性死亡,并且可通过伤口和呼吸道等传播途径,导致人的感染发病和死亡。1998年和2005年在我国江苏"苏中"地区和四川资阳等市县人群中曾先后两次暴发大规模SS2感染人的事件。人感染病例中出现了从未报道过的链球菌中毒性休克综合征(Streptococcal toxic shock syndrome,STSS),病死率高达80%以上,已成为重要的新发传染病病原体[1-4]。     

Identification and characterization of novel immunogenic proteins of Streptococcus suis serotype 2

Streptococcus suis, a zoonotic pathogen, caused serious outbreaks in humans with high mortality rates in the past decade. To develop safer and more effective vaccines, particularly for human protection, cell wall and extracellular proteins of S. suis serotype 2 were analyzed by an immunoproteomic approach in this study. Thirty-two proteins with high immunogenicity were identified and 22 of them were newly identified. Further analyses of 9 selected proteins revealed that (1) these 9 proteins were expressed in all tested virulent S. suis serotype 2 isolates, (2) antisera against 6 of the selected proteins efficiently killed the bacteria by opsonized phagocytosis in human blood, and (3) significantly higher levels of serum antibodies against 3 proteins were detected in both patients and infected swines. Therefore, our results suggest the 3 proteins (SSU98_0197, SSU98_1094 and SSU1664) have strong potential to be vaccine candidates.     

猪链球菌2型89K毒力岛研究进展

高致病性猪链球菌2型的致病机制仍是未解之谜.毒力岛不仅赋予病原菌特殊的致病能力,而且在细菌的适应性进化过程中扮演重要角色.对猪链球菌2型89K毒力岛功能性基因的深入剖析有助于更全面地掌握病原菌的致病特性.综述了猪链球菌2型89K毒力岛的结构与进化过程,以及国内外对毒力岛中二元信号转导系统、Ⅳ型分泌系统、ABC转运蛋白、毒素-抗毒素系统等重要基因的研究进展,力图从基因水平为猪链球菌2型的致病机制寻找突破口.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Simple broad-spectrum protocol using labiase for bacterial cell lysis to prepare genomic DNA for pulsed-field gel electrophoresis analysis

We have developed a simple broad-spectrum protocol using labiase for bacterial cell lysis in pulsed-field gel electrophoresis analysis. The protocol reported here is widely applicable to the preparations of genomic DNA from Gram-negative and -positive pathogens, including enterococcal strains resistant to any conventional lysis protocols.