| Streptococcus suis (S. suis) is an important pathogen associated with a wide range of disease in pigs, including meningitis, septicaemia, pneumonia, endocarditis, and arthritis. Thirty-three serotypes (types 1-31,33, and 1/2) have been described based on capsular polysaccharides. Between July and August 2005, an outbreak of S. suis type 2 infection occurred in Sichuan, China, involving 200 cases, of whom 38 died, which highlight the importance of Streptococcus suis in public health. In this study, we analyzed the proportion of Streptococcus suis in swine Streptococcal disease isolates, the genotype of virulence associated factors and the relationship between the virulence and these traits. Fibrinogen is the main component in the swine plasma and it could induce the biofilm formation of Streptococcus suis. As biofilm formation by pathogenic microorganisms was a mechanism that allows them to become persistent colonizers, resist clearance by the innate and adaptive host immune system, enhance their resistance to antibiotics, and exchange genetic material. In this study, we found that the biofilm producer only had lowly virulence compared to the strains with the same genotype. So we used the biochip to compare the transcripts of Streptococcus suis cultured in broth with and without fibrinogen. During the up-regulated genes, we selected two genes to further determine their roles in biofilm formation and pathogenesis of Streptococcus suis. The principal results were described as the following:1. The epidemiology of Streptococcus suisThrough the systemic epidemiological investigation, we found that 407 (56.6%) were identified as S. suis strains during the 719 Streptococci isolates from 2003 to 2007 and it had substituted Streptococcus equi subsp. zooepidemicus prevailing in China. During the isolates, the serotype 2 was most frequent (43.2%) which followed by the serotype 3 (14.7%),4 (6.4%),8 (4.7%),5 (3.9%),7 (3.7%), 1/2(3.2%),16 (2.9%), and 17 (2.5%). Serotype 2 was significantly associated with the systemic infection, whereas, serotype 3 was more frequently isolated from swine with pneumonia.54% of the serotype 2 strains were with the genotype of sly+mrp+epf+ which were well known to be highly virulent through the mice infection test. Ongoing survey from 2008 to 2009, serotype 2 was the main serotype with either systemic infection or pneumonia. The importance of serotype 3 during pneumonia had decreased. The proportion of serotype 21 was higher than that from 2003 to 2007. Serotype 2 had become the main serotype responsible for swine Streptococcal disease. Through the determination of tetracycline resistance gene and multilocus sequence type, we found that there were only 6 isolates were all ST7- Tn916- tetM lacking tetO clone with the genotype of sly+mrp+epf+, indicating that the highly virulent outbreak clone only sporadic existed in the swine population in China. But to our concern, most of isolates were included in ST1 and ST27 complex, which represent the highly virulence clone complex. And there were many ST1 and ST7 clone included which continued evolving. The strain ST and their ability to confer swine blood cell phagocytosis were closely related. The ST1 strains were mostly able to confer phagocytosis, while ST7, ST27 complex strains were in the middle, other ST related strains were worst. Because the MLST scheme was time consuming, in order to overcome this issue, we developed a rapid and high-throughput S. suis multilocus sequence typing (MLST) approach based on comparative sequencing by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The whole process was only 6.5 hours. This high-throuput automated method appears to be an ideal tool for real-time investigation of S. suis outbreaks.2. The application of biofilm formation strainDuring the clinical Streptococcus suis isolates, we found a weak virulent strain with biofilm formation ability belongs to serotype 2 with the genotype sly+mrp+epf+. The pigs immuned with this strain could confer highly virulent strain ZYS19 lethal infection. But the safety dose of this strain for pigs was 5×107CFU, which was not safe enough in the clinical application. In order to maintain its immunogenicity and increase the safety, we constructed the aroC gene mutant of this strain. The result showed that:the mutant strain grew slower compared to the wild type strain in THB broth and the growth rate was repaired when the broth was added with aromatic amino acids. In order to assess the safety and protective efficiency, we immuned the pigs with 108CFU and 109CFU each 4 pigs twice. The whole process was without body temperature elevated. Two weeks after second immunity, the antibody against the capsular antigen in the sera increased. When attack with highly virulent clone ZYS19 at the same time, the pigs in the control group all died, whereas,50% of the pigs in the immunity group with 108CFU survived and 75% of the pigs immuned with 109CFU were protected. Hematoxylin-eosin staining of various tissue samples showed that the pigs in the control group showed neuronal necrosis, myocardial hemorrhage, spleen necrosis. But the pigs in the immune group were normal. Although the immune group showed minor atelectasis, it was less serious than the control group.3. The mechanism of biofilm formationIn this study, biochip was used to compare the transcripts of the biofilm and planktonic cells.(1)The results showed that there were 648 differential transcripts with 330 genes downregulated and 318 genes upregulated. Among these regulated genes, those associated with protein synthesis, DNA replication, sugar transport and metabolism were down regulated, which indicated that the metabolic activity of cells in the biofilm was overall reduced. Whereas, genes associated with cell wall and capsular synthesis were downregulated and that with pilus export and sortase, hemolysin were upregualted, which could help S. suis to establish infection.(2)LeuD and autolysin were selected which upregulated a lot for further research. Through real-time PCR, these two genes were all upregualted during biofilm formation, which indicated that they were closed related with biofilm formation. The mutant of these two gene showed reduced ability of biofilm formation, adherence to Hep-2 cells and toxicity to SJPLC cells. In addition, these two mutant had the lower ability to confer swine blood cell phagocytosis and decreased virulence to mice. |
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