| Viridans Streptococci(VS), as resident flora of mouth, upper respiratory tract and gastrointestinal tract, is previously thought to be a part of the body’s normal flora. Recently, cases of oral and maxillofacial soft tissue, reproductive tract infections and endocarditis caused by VS, are increasing. Infective endocarditis, most common systemic infection caused by Viridans Streptococci, is thought to be generated by transient bacteremia after dental treatment, even teeth brushing. PS also can trigger human immune response, and is an important source of infection in patients with cancer, bone marrow transplantation, immune deficiencies such as pneumonia. Septicemia caused by VS infection often cause septic shock leading to death. Now VS is regarded as one of odontogenic infection common pathogens, and involve in the deep abscess formation. Correlation studys between periodontitis and coronary heart disease show that VS can promote platelet aggregation, accelerate blood clotting, thrombosis, which may lead to the low bloody cardiovascular disease. Early atherosclerotic damage, acute coronary syndrome or ischemic stroke correlated to VS have be reported. It has be reported antimicrobial susceptibility of VS significantly cut down, where penicillin resistance rate is 22.4%, ciprofloxacin, tetracycline, clindamycin, sulfamethoxazole, erythromycin resistance rates is at 60% -80%.More than 90% of the natural bacteria live in the form of bacterial biofilm (BF), and more than 65% of human bacterial infections are related to BF. Through a variety of mechanisms, BF play an important role in barrier protection to prevent or reduce dispose bacteria by drug and immune system, which significantly increase virulence and resistance of BF. It has been confirmed that VS is able to form biofilms, which is an important factor of VS resistance. Biofilm bacterial resistance mechanisms include mechanism of biofilm barrier, nutrient limitation, P-lactamase and quorum sensing.Quorum sensing (QS) is widespread in gram negative and positive bacteria, which is a density -dependent gene expression and regulation mechanism. Bacteria produce and secrete autoinducer (AI) into the extracellular environment, when the AI signal concentration reaches the threshold value, AI molecules bind to bacterial surface receptor protein, and then directly or indirectly regulate expression of bacterial gene. The formation and stability of BF, virulence factor expression, acid and drug resistance are regulated by AI signaling molecules, which is important to the establishment of infection. It’s a focus that reduce the difficulty of antibiotic treatment or naturaly eliminate the infection by host defense system, which is via reducing virulence and resistance of biofilm when quorum sensing is interfered.QS common regulatory mechanisms can be divided into three types:Lsr, AI-1 and AI-2. Lsr and AI-1 mediate bacterial intraspecific communication, AI-2 is a general signal molecules of bacterial interspecific communication. The interspecific exchange under control of AI-2 has been confirmed to play an important role in the process of biofilm formation, development and functioning.LuxS gene is considered to be iconic gene about AI-2 signaling molecule synthesis, because the protease encoding by LuxS gene is necessary for AI-2 synthesis. Researchers have confirmed LuxS gene mutation can interfere with AⅠ-2, and then affect biofilm formation, drug and acid resistance.In this study, by knocking out LuxS gene of Viridans Streptococci wild strain with multidrug resistance, blocking AI-2 synthesis, disturbing quorum sensing, affecting biofilm formation and resistance are carried out. It’s the purpose to discuss what the role of LuxS/AⅠ-2 quorum sensing signal in Viridans Streptococci biofilm formation, ampicillin, quinolones and tetracyclines resistance is, and to find a new target to reduce bacterial virulence.Chapter OneIsolation and Identification of Viridans Streptococci with Multidrug ResistanceObjective To get a target strain from clinic, which is Viridans Streptococci with multidrug resistance. identify species of Viridans Sstreptococci with multidrug resistance. Methods Culture in blood plate; Gram stain and susceptibility test were first carried out. After preliminary screening and pure culture, physiological and biochemical tests; 16S rDNA sequence homology analysis:the genomic DNA of the bacteri was extracted, and 16S rDNA was amplified by using polymerase chain reaction. Then 16S rDNA sequence was analyse and compared. Results Clinical isolate No.327045 with multidrug resistence is, Gram positive streptococcus, alpha haemolysis. It’s mostly possible to be D group of streptococcus, S. mitis, according to biochemical tests. The result of 16S rDNA sequence homology analysis suggests that isolate No.327045 is highly matched with S. mitis,99.58%. Conclusion Clinical isolate No.327045 is Viridans Streptococci with multidrug resistance. It’s the target strain.Chapter TwoVirulence Detection of Viridans Streptococci with Multidrug ResistanceObjective To detect the virulence of Viridans Streptococci, and discuss the mechanism. Methods The autoinducer-2 (AⅠ-2) bioluminescence assay of Vibrio Harvey BB170 strain, multidrug resistance, ability of biofilm formation was examined in Viridans Streptococci wild strain biofilm. The structure, exopolysaccharides and dead/live cells of biofilm were scaned by Confocal laser scanning microscope (CLSM). Susceptibility test was experimented. Results Viridans Streptococci wild strain formed biofilm. It’s structure is complex and disproportion. Antibiotic can affect biofilm formation. Conclusion Viridans Streptococci with multidrug resistance can form numerous biofilm and be high virulence.Chapter ThreeConstruction of Viridans Streptococci LuxS Gene MutantObjective To construct a LuxS gene mutant of Viridans Streptococci, by knocking out the entire LuxS gene of Viridans Streptococci strain with multidrug resistance via homologous recombination. It’s a tool to studying the function of LuxS gene in biofilm formation and multidrug resistance. Methods Erythromycin resistance gene, the upstream and downstream of LuxS were cloned respectively by using plasmid PMG36E and DNA of S. viridansas template. Then the genes were ligated into Multiple Cloning Site (MCS) of vector pUC19 in certain order and transformed into E. coli competent cells. Finally transformants were selected for resistance to erythromycin and ampicillin. Electrotransformation of Viridans Streptococci cells with pUCluxKO—mutant resulted in isolation of erythromycin resistant Viridans Streptococci transformants, which was identified by polymerase chain reaction, Results PCR agarose gel electrophoretic analysis suggested that erythromycin resistance gene, the upstream and downstream of LuxS were successfully ligated into accurate enzyme digestion site of vector pUC19, and LuxS gene was completely replaced by erythromycin resistance gene in Viridans Streptococci mutant. Conclusion Viridans Streptococci LuxS gene mutant was successfully constructed.Chapter FourInfluence of LuxS gene deletion on Viridans Streptococci VirulenceObjective To study the influence of LuxS gene of Viridans Streptococci on biofilm virulence, and discuss the mechanism. Methods The autoinducer-2 (AI-2) bioluminescence assay of Vibrio Harvey BB170 strain, multidrug resistance, ability of biofilm formation was examined in LuxS mutant and wild strain. The structure, exopolysaccharides and dead/live cells of biofilm were scaned by Confocal laser scanning microscope (CLSM). Compared with each other. LuxS mutant growth compensated by wild strain supernant or DPD was experimented. Results LuxS gene deletion decreased VS wild strain biofilm formation, loosen structure and changed multidrug resistance. This is because of LuxS gene deletion, AI-2 signaling moleculer dyssynthesis and bacteria communication outage. Conclusion LuxS gene/ AI-2 signal can influence the virulence of Viridans Streptococci biofilm with multidrug resistance. It’s a new target to treat the bacterial biofilm infection. |
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