ABSTRACT
Objective To study the biological characteristics of clinical isolates of human infected Streptococcus suis type 2 in Zhongshan City from 2016 to 2019 and classify the strains using pulse-field gel electrophoresis (PFGE), and to provide a scientific basis for clinical prevention and treatment of porcine streptococcus suis disease. Methods Twelve strains of Streptococcus suis type 2 were collected from 2016 to 2019 and identified by automatic bacterial identification instrument. The carrying status of five major virulence genes of Streptococcus suis was detected by nucleic acid and protein analyzer, including capsular polysaccharide (cps2J), lysozyme-releasing protein (mrp), hemolysin (sly), glutamate dehydrogenase (gdh), and extracellular factor (ef). The susceptibility of Streptococcus suis to 12 kinds of commonly used antibiotics was determined by the broth microdilution method, and the homology analysis was carried out by PFGE method. Results Twelve strains of Streptococcus suis type 2 were divided into four virulence genotypes, mainly mrp-/sly+/ef+/cps2J+/gdh+ (6strains) and mrp-/sly-/ef+/cps2J+/gdh+(4strains). Drug susceptibility test results showed that 12 strains of Streptococcus suis type 2 were resistant to erythromycin, tetracycline and clindamycin, and they all were multi-resistant strains. According to the classification results of PFGE, the 12 strains were classified into 7 PFGE types based on 100% similarity coefficient. The PFGE band types of Streptococcus suis in the same year had high homology. Conclusion The virulence genotypes of 12 clinical isolates of human infected type 2 Streptococcus suis in Zhongshan from 2016 to 2019 are diverse, and the strains are resistant to multiple antibiotics. Most strains in the same year are the same clone strains. PFGE genotypes are not correlated with virulence genotypes and drug resistance spectrum.
ABSTRACT
Objective To investigate and analyze the epidemiological and pathogenic characteristics on a case of human Streptococcus suis type 2 infection in Minhang District, Shanghai, and to provide evidence for early warning and prevention and control measures of rare and imported zoonotic acute infectious diseases in Shanghai. Methods By inquiring the patient medical history and epidemiological history and on-site environmental investigation, the infection route and source of the case were examined. The pathogenic culture of cerebrospinal fluid (CSF) was used to isolate Streptococcus suis, and Vitek2GP was used to identify the isolated strains. The PCR technique was used to detect species specific genes and virulence genes. Results The clinical manifestations of the patient were high fever with headache, nausea, vomiting and stiff neck. Blood tests showed a significant increase in c-reactive protein, an increase in lymphocyte percentage, and a decrease in platelet count. Head CT examination showed bilateral ethmoidal sinus and bilateral maxillary sinus inflammation, and significantly increased CSF white blood cell count and immunoglobulin. The case's CSF sample was positive for species specific genes (16SrRNA) and 2 virulence genes (cps-2j and ef). Conclusion This case was human Streptococcus suis type 2 with meningitis symptoms. Good prognosis was associated with timely diagnosis and treatment as well as the types of virulence factors. Medical institutions should identify early infection and take timely treatment as soon as possible to avoid severe illness and death cases. Departments of agriculture, health, market management, and others should consummate the reporting mechanism of animal epidemic situation, and establish necessary active sentinel monitoring.
ABSTRACT
Aims: The objective of this study is to identify S. suis type 2 and evaluate the virulence of ZHJ01 strain isolation, and verity the clinical and pathological outcome of a systemic infection caused by one serotype 2 when simultaneously inoculated with ZHJ01 strain. We also want to clarify the epidemiologic, clinical, microbiologic characteristics and the pathogenesis mechanism of S. suis type 2 in Hubei province, China. Study Design: Pigs suspected of being infected with S. suis in Jingzhou regions of Hubei province, China were studied. S. suis type 2 isolation was obtained from the suspicion of diseased pig. The case of S. suis type 2 was detected by the virulence factor amplification based on PCR detection and bacterial isolation, identification in the laboratory. According to the experimental infections of mice and piglets, pathogenicity of this S. suis type 2 isolation to mice and swine was monitored. This study was conducted in the key laboratory of pathogenic microbiology, College of Animal Science of Yangtze University, and Institute of Black Pigs Research, Yangtze University. Methodology: Proper serological typing can be performed using a co-agglutination test. The typical colonies purificated and cultured were inoculated with Glucose, Lactose, Raffinos, Sorbitol, D (+)-sucrose, Trehalose, 6.5%NaCl, D (-)-Salicin, Hippurate, Esculin hydrate, V-p, etc., then the test results were recorded. Detection of virulence factors were performed using PCR amplification and DNA sequencing. S.suis type 2 isolation was inoculated to mice and piglets for the virulence test, and the observation of the clinical signs and pathological changes. Results: The virulence factor of extracellular protein factor (EF) was determined from ZHJ01 strain based on PCR detection. Sequence analysis indicated that the isolate was very similar to nucleotide homology with others SS2 strains from different county or contries, and there was not much variation. LD50 of S. suis type 2 for mice was 2.5 x 107cfu. LD50 of S. suis type 2 for piglets was 3.92 x 109cfu. Conclusion: The results show that Swine S. suis type 2 has a relatively strong pathogenicity to pigs in Hubei province, China. This study can be, in part, sufficient to explain the pathogenicity for ZHJ01 strain in area of Zhijing, Jingzhou city, China, which may provide insights into the pathogenesis SS2 and more valid data to support the development of S. suis vaccine as well as the epidemiological investigation, further monitoring and effective prevention to S. suis.
ABSTRACT
Objective@#To construct the mutant strain ATP binding cassette transporter SSU05_0948 of Streptococcus suis type 2 and comprehensively study its pathogenicity, and to provide useful insights for understanding the mechanism that Streptococcus suis avoid host innate immunity. @*Methods@#The mutant strain 05ZYH33Δ0948 was constructed through homologous recombination technology. The differences between the mutant strain and the wild type strain were evaluated through bacterial adhesion, whole blood killing, mice meningitis assay, mice and piglets virulence assay. Chi-square test and t test were used. @*Results@#Successfully constructed the mutant strain 05ZYH33Δ0948. The adhesion results showed that the adhesion rate (0.663±0.047)% of the wild strain to A549 cell was significantly higher than that of the mutant (0.246±0.074)%, the difference was statistically significant (χ2=5.267, P=0.014); the adhesion rate (16.540±2.320)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.970±0.320)%, the difference was statistically significant (χ2=0.014, P<0.01); the adhesion rate (5.497±0.174)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.950±0.335)%, the difference was statistically significant (χ2=0.016, P<0.01). The killing rate (32.970±3.589)% of the wild strain in whole blood is no difference with the mutant (29.560±3.737)% (χ2=1.200, P=0.133). Piglets competitive infection showed that, the competitive index at 12 h, 24 h and 36 h were 0.046±0.003, 0.107±0.003, 0.064±0.001, respectively. 12 h and 24 h was significant differences(t=15.490, P=0.041), 24 h and 36 h was significant differences(t=5.660, P=0.047), 12 h and 36 h was no differences(t=1.445, P=0.285). @*Conclusions@#Streptococcus suis type 2 ABC transporter SSU05_0948 is a new adhesion factor and virulence factor of Streptococcus suistype 2, and also a new meningitis factor, which plays important roles in Streptococcus suis against host innate immunity.
ABSTRACT
Objective Serine /threonine kinases (STK) and phosphatases (STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins .This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2, the Chinese virulent strain 05ZYH33. Methods The double mutant of the stk and stp1 genes of 05ZYH33 was constructed by homologous recombination .The biological characteristics of the wild strain 05ZYH33 and the mutant strain Δstk/stp1 were compared.The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay , anti-phagocytosis assay and the mouse model of infection . Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spc r and the mutant strain was successfully constructed .Experi-ments of biological characterization revealed gradually increased value of 05ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours.The logarithmic phase of the mutant strain (A600≈0.4) was 1 hour later than that of the wild one , and the bacterial den-sity of the former was lower than that of the latter after the plateau pe -riod (0.8 vs 1.0).On the blood plates of 05ZYH33 and Δstk/stp1 were observed greyish, round, semitransparent, wet and smooth-sur-faced tiny bacterial colonies , around which there were hemolysis rings with no significant differences in colony morphology and hemolytic ac -tivity.In the experiment on pathogenicity , the mice of the 05ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours. Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacte -rial proliferation and division.
ABSTRACT
Objective To study the function of gene 0955 in Streptococcus suis type 2 98HAH33. Methods Growth condition of wild-type, mutant and complemented strains of Streptococcus suis type 2 98HAH33 was compared at different stages. Differences in adhesion ability to host cells and anti-phagocyto-sis among these strains were compared by using bacterial adhesion test and analyzing their survival rates in blood. Mouse and piglet models were used to evaluate their virulence. Results The growth of the mutant and the complemented strains was slightly slower than that of the wild type strains in logarithmic growth phase, but no significant difference was found in plateau phase. Bacterial adhesion test showed that gene 0955 might encode a new adhesion factor of Streptococcus suis type 2 98HAH33. Blood bactericidal test sug-gested that gene 0955 was not associated with anti-phygocytosis. Animal experiments showed that gene 0955 might be a novel virulence gene of Streptococcus suis type 2 98HAH33. Conclusion Gene 0955 might en-code a novel adhesion factor and virulence factor of Streptococcus suis type 2 98HAH33.
ABSTRACT
Objective To construct a mutant strain of Streptococcus suis type 2 05ZYH33 express-ing ABC transporter SSU05 0946 and to study the pathogenicity of ABC transporter SSU05 0946 for better understanding the immune evasion strategies by Streptococcus suis. Methods Genome of the Streptococcus suis type 2 05ZYH33 strain was extracted and used as a template to amplify SSU05 0946 upstream and down-stream homeodomains. Chloramphenicol-resistance gene was amplified by using pSET1 plasmid as the tem-plate. These three amplified fragments were fused and integrated with the thermo-sensitive plasmid pSET4s by using overlap extension PCR. Homologous recombination method was used to construct the mutant strain 05ZYH33Δ0946. Differences between the mutant and wild type strains were evaluated through bacterial ad-hesion assay,whole blood killing assay and challenge test in mice and piglets. Results The mutant strain 05ZYH33Δ0946 was successfully constructed. Results of bacterial adhesion assay demonstrated that SSU05 0946 was not involved in the adherence of Streptococcus suis to human epithelial cells. SSU05 0946 was an ovel anti-phagocytic factor and virulence factor of Streptococcus suis. Conclusion Streptococcus suis type 2 ABC transporter SSU05 0946 is a newly discovered virulence factor of Streptococcus suis, playing an impor-tant role in the evasion of host innate immunity by Streptococcus suis.
ABSTRACT
Streptococcus suis is regarded as one of the major pathogens of pigs, and Streptococcus suis type 2 (SS2) is considered a zoonotic bacterium based on its ability to cause meningitis and streptococcal toxic shock-like syndrome in humans. Many bacterial species contain genes encoding serine/threonine protein phosphatases (STPs) responsible for dephosphorylation of their substrates in a single reaction step. This study investigated the role of stp1 in the pathogenesis of SS2. An isogenic stp1 mutant (Δstp1) was constructed from SS2 strain ZJ081101. The Δstp1 mutant exhibited a significant increase in adhesion to HEp-2 and bEnd.3 cells as well as increased survival in RAW264.7 cells, as compared to the parent strain. Increased survival in macrophage cells might be related to resistance to reactive oxygen species since the Δstp1 mutant was more resistant than its parent strain to paraquat-induced oxidative stress. However, compared to parent strain virulence, deletion of stp1 significantly attenuated virulence of SS2 in mice, as shown by the nearly double lethal dose 50 value and the lower bacterial load in organs and blood in the murine model. We conclude that Stp1 has an essential role in SS2 virulence.
Subject(s)
Animals , Humans , Mice , Bacterial Load , Lethal Dose 50 , Macrophages , Meningitis , Oxidative Stress , Parents , Phosphoprotein Phosphatases , Reactive Oxygen Species , Streptococcus suis , Streptococcus , Swine , VirulenceABSTRACT
Streptococcus suis é um patógeno que a afeta a produção industrial de suínos em todo o mundo. É de extrema importância, pois está associado a doenças em suínos e humanos. O objetivo deste estudo foi determinar a prevalência do Streptococcus suis tipo 2 em 201 amostras de tonsilas de animais clinicamente sadios a partir da técnica de PCR. As amostras positivas foram submetidas à pesquisa do gene codificador do fator extracelular (ef). Os resultados demonstraram que a prevalência (23,38 por cento) foi maior que em outro estudo recentemente realizado no mesmo Estado, indicando que a PCR é um método mais sensível em relação ao isolamento bacteriano. Houve baixa ocorrência do gene ef* (1,49 por cento), o que mostra uma grande importância para população analisada, pois cepas negativas são potencialmente menos virulentas que cepas positivas.
Streptococcus suis is a pathogen that affects the industrial production of swine worldwide. It is extremely important, because it is associated with pigs and humans diseases. The aim of this study was to determine the prevalence of Streptococcus suis type 2 in 201 samples of tonsils from clinically healthy animals by the PCR technique. The samples positive for S. suis type 2 were tested for the gene encoding extracellular factors (ef). The results showed that the prevalence (23.38 percent) was higher than other recent survey in the State, demonstrating that the PCR is a more sensitive method in relation to the bacterial isolation. There was a low occurrence of ef* gene in samples (1.49 percent) showing great importance to local swine population, because negative strains are potentially less virulent that positive strains.
ABSTRACT
To investigate the distribution of virulence associated factors of 5 strains of Streptococcus suis type 2 from slaughtered pigs isolated in Fujian province , 7 virulence genes were detected through PCR amplification. They were the glutamate dehydrogenase gene(gdh), the capsular polysaccharides gene(cps), the extracellular factor gene(ef), the muramidate-released protein gene(mrp), the suilysin gene(sly), the fibronectin-binding protein gene(fbps) and the virulence-associated sequence or f2. Among these 5 strains isolated, the virulence genotypes of 4 strains S.suis type 2 and strain SS2PFJ07 was gdh+/cps2J+/mrp+/ef-/sly-/fbps+/orf2+ and the other one strain isolated from slaughtered pigs was gdh+/cps2J+/mrp+/ef+/sly+/fbps+ /orf2+ respectively. This results showed that there are at least 2 virulence genotypes in Streptococcus suis type 2 from slaughtered pigs isolated in Fujian province.
ABSTRACT
To identify the cell wall-binding proteins of Streptococcus suis serotype 2(SS2), according to their structure feature, the substrates of sortase and I type secreted proteins with LysM domain, WxL domain, choline-binding domain,GW domain or S layer homologous domain in the recently published genome of SS2 strain 98HAH33 were firstly identified and then the putative functions were attributed to individual proteins by reference to the identification of conserved domains of InterPro and BlastP servers. Homologous proteins were identified by unfiltered BlastP homology searches (including conserved domain detection). Among the 23 putative proteins with a C-terminal LPXTG recognition signal for covalent attachment to peptidoglycan by sortase, 9 with I signal peptide were identified as sortase substrates. Among 9 substrates , YP_001201232, YP_001201531 and YP_001201656 had been experimentally verified to anchor to bacterial cell wall , and YP_001201232 known as the opacity factor of S. suis (OFS) was proved to be the virulence factors. According to function analysis, YP_001201484, YP_001201544, YP_001199825, YP_001197640, YP_001197840 and YP_001199755 appeared to be involved in SS2 pathogenesis. and YP_001200959, YP_001201233 and two proteins with LysM domain( YP_001199784 and YP_001201729) were the hypothetical proteins. These data suggest the majority of putative sortase substrates may implicate in the virulence of SS2 and could serve as a basis for targeted experimental studies into the function of these proteins.
ABSTRACT
The hemolysin production of 19 strains of Streptococcus spp. isolated from pigs in the Jiangsu and Shanghai regions in recent years were examined. Eight isolates of Streptococcus suis type 2 from Jiangsu showed weak hemolysis on blood agar, but a stronger reaction in Todd-Hewitt broth(THB). The hemolysin belonged to the group of thiol-activated hemolysins. Nine strains of Streptoccus equi subsp. zooepidemicus showed strong hemolysis on blood agar and in THB containing 5% new-born calf serum, but no hemolysis in THB alone. This hemolysin was similar to streptolysin S(SLS). Another two isolates were atypical members of Lancefield group C Streptococcus and showed strong hemolysis on blood agar and in THB with 5% new-born calf serum, but the hemolysin was unlike either streptolysin O or SLS.
ABSTRACT
To construct gene knock-out mutant of a two-component signal transduction system named 2148hk/rr in Streptococcus suis type 2 virulent strain 05ZYH33.Recombinant gene knock-out vector was constructed consisting of Spc~r cassette with flanking homology regions to the target genes,the isogenic 2148hk/rr-deficient mutant was screened by allelic replacement.PCR analysis and Southern hybridization confirmed that the coding genes of 2148hk/rr were replaced completely by spc~r cassette.Conclusion The mutant of 05ZYH33 2148hk/rr system was successfully constructed,which laid the foundation for farther research on the role of this two-component signal transduction system during infection.