| Streptococcus suis is an important pathogen of swine. It causes septicemia, endocarditis, meningitis, arthritis, pneumonia and other life threatening diseases. At present, domestic and foreign scholars think that Streptococcus suis can form biofilms in vitro. Bacterial biofilm attached to the solid surface with micro structure of the bacterial community is the secretion of aqueous polymer matrix. Streptococcus suis biofilm formation provides a protective way for the bacteria to improve the resistance and pathogenicity.Biofilm affected by many factors and regulation is an important way of life. The quorum sensing(QS) is one of the factors of phenomenon, in recent years, and it is discovered on the biofilm and is important effect. Gram positive bacteria QS system is mainly used for small molecular peptides or AIP(autoinducter peptide) as inducer. The AIP molecules of different bacteria have different sizes, and they can not free to penetrate the cell wall. The AIP molecules reach the extracellular function by the ABC transport system(ATP-Binding-Cassette) or the membrane channel protein. By the quorum sensing system, the bacteria can coordinate to complete a series of life activities, such as bioluminescence, synthesis of protease, virulence factors, and promote the agglomeration between the cell body and phenotypic changes.The study found that quorum sensing system and bacterial biofilms are closely related.Some recent studies suggest that some antibiotics are difficult to remove biofilm bacteria, but at sub inhibitory concentrations(MICs) can form biofilm intervention. The antibiotics provides hope for the effective prevention of clinical bacterial biofilm formation. In view of the fact which has a good prospect for clinical application of antibiotics, sub-MIC antibiotics began to pay attention to the role of bacterial biofilm formation. In vitro, the concentration of sub-MIC macrolides showed inhibition of biofilm formation by Pseudomonas aeruginosa and Mycobacterium avium. But it not yet have an impact on the study of sub-MIC macrolide antibiotics against Streptococcus suis biofilm formation and regulatory mechanisms. In the present study, we obtain the success of Streptococcus suis in vitro biofilm by using the Calgary biofilm device(CBD).We use qualitative and quantitative crystal violet staining method and scanning electron microscope method to study the effect on biofilm of Streptococcus suis ATCC700794 on micro plate three macrolides(azithromycin, erythromycin, tylosin streptozotocin). Using i TRAQ technology in 1/4MIC by proteomics, macrolides(azithromycin, erythromycin, tylosin) quantitative proteome analysis of biofilm formation of Streptococcus suis intervention under the effect of the concentration. We explore the relationship between quorum sensing system and biofilm formation of Streptococcus suis. By Real time PCR technology, virulence factors genes and quorum sensing proteins are changed. We find that the macrolide drug intervention target for biofilm formation of Streptococcus suis is effect. We study the possible mechanism of quorum sensing system, and the relationship between the expression of biofilm formation and virulence gene. The main research results are as follows:(1) Through the Calgary biofilm device, in vitro biofilm of Streptococcus suis can form, and the biofilm formation mature period is 72 h.(2) Sub-MIC of macrolides(azithromycin, erythromycin, tylosin) affect Streptococcus suis biofilm formation by crystal violet staining of the biofilm quantitative analysis, which show that in the 1/2 and 1/4MIC concentration affect Streptococcus suis biofilm formation.(3) By proteomic technology of i TRAQ, the concentration of 1/4MIC of macrolides(azithromycin, erythromycin, tylosin) affect Streptococcus suis by quantitative proteome analysis of biological membrane. According to the ratio >1.2 or <0.8(p-value <0.05) protein screening, results showed that erythromycin intervention, 55 up-regulated proteins, down protein 24; Azithromycin intervention, 82 up-regulated proteins, 92 down regulated proteins; tylosin intervention, 105 up-regulated proteins, 66 down regulated proteins. The quorum sensing system in the ABC superfamily ATP binding cassette transporter protein(gene comAB) were up-regulated.(4) Through Real time PCR method, 1/4MC erythromycin and azithromycin, tylosin intervention related gene of Streptococcus suis and virulence genes of QS system under the com AB are studied. On the sub-MIC of erythromycin of Streptococcus suis biofilm formation, GDH, FBPS, EF expression is the up-regulated. Com AB gene and virulence in QS system and CPS expression was down regulated, and other genes expression was no significant difference. Sub-MIC of azithromycin affect Streptococcus suis biofilm formation, CPS, MRP and EF expression is the up-regulated. ComAB gene, virulence in QS system, GDH and sly expression were down regulated, other genes expression was no significant difference. Sub-MIC tylosin inhibits biofilm formation of Streptococcus suis, the expression of comAB in QS system, the virulence genes of GDH, sly, CPS, GAPDH, FBPS, MRP and ef were up-regulated expression. Through the QS system, the drug may be involved in the regulation of virulence gene expression.The results of this study suggest that macrolide antibiotics in the sub-MIC concentration can affect on biofilm formation of Streptococcus suis ATCC700794. The mechanism may be that sub-MIC macrolide drugs act on the quorum sensing system in the ABC superfamily ATP binding cassette transporter protein, and then affect biofilm formation and virulence gene expression.Using sub-MIC antibiotics to control bacterial biofilm is poorly understood, and there are still many unknown. Therefore, a large number of related laboratory research can draw on the advantages and avoid disadvantages in use, which provide valuable reference for the reasonable application of the research and which also can use the bacterial biofilm related regulation mechanism to more effectively control the bacterial biofilm infection. |
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