LuxS基因缺失对变异链球菌生物学性状、生物被膜结构的影响

发布时间：2018-03-08 22:37

本文选题：变异链球菌　切入点：LuxS基因　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：本实验是通过采用常规生化鉴定、革兰氏染色涂片鉴定和生长曲线测定等方法阐述了变链菌标准株与LuxS基因突变株在生物学性状方面的异同。此外,建立LuxS基因缺失突变株的生物被膜体外模型,用结晶紫染色等方法观察变链菌生物被膜结构,研究LuxS基因突变对其形成生物被膜的能力的影响,并借助于扫描电镜观察了LuxS基因缺失突变株和标准株所形成的生物被膜,为进一步研究LuxS信号系统对变链菌致龋毒力的调控机制奠定基础。方法：将变链菌标准株与LuxS突变株分别在含红霉素及不含红霉素的TSA固体培养基中作菌落培养,观察两种菌落的生长情况并作常规生化检测、革兰氏染色涂片以比较两种菌株的生长特性。将复苏24h的标准株及LuxS突变株于紫外分光光度计600nm处制备成吸光度A=1.0的菌悬液备用。将上述菌悬液1000倍稀释接种于BHI液体培养基,置于37℃恒温摇床中孵育,定时取样测量A值,观察比较两者在生长曲线上的差异。将LuxS基因突变株和标准株接种于BHI液体培养基中,培养48h后,取出相应的微孔板,结晶紫染色生物被膜,观察细菌生物被膜的形态变化。将提前制备好的无菌牙釉质磨片置于6孔细胞培养板中,并依次编号第1-6孔,每孔放置4片,分别取变链菌标准株和LuxS突变株的A600=1.0的菌悬液向每孔中接种1ml(其中第1、4孔接种菌液为标准株,2、3、5、6四孔接种菌液为LuxS突变株),均匀滴至牙釉质磨片表面,静置约2min后向每孔中加入5mlTPY液体培养基(其中第1、2、3孔为含2%葡萄糖的TPY溶液,4、5、6孔为含2%蔗糖的TPY溶液),然后取提前制备好的标准株上清液各500μl加入至3、6两孔。将上述菌液于37℃厌氧条件下培养24h,于扫描电镜下观察在无菌牙釉质磨片上形成的生物被膜,并进行总体评价以对比两种菌株的生物被膜的形成能力。结果：在含红霉素的TSA-Eymr固体培养基中,标准株基本不能生长,而突变株的生长状况基本正常。在不含红霉素的TSA固体培养基中培养,两种菌株的菌落形态没有明显差别,镜下可见标准株菌体呈长链状排列且相互缠绕,而突变株菌体多呈短链状排列,形成长链的较少。通过对变链菌标准株与LuxS突变株测定生长曲线后发现,两者在生长模式上并无明显差异,只是在进入生长的稳定期后,两者在细菌饱和度上有一定差异,标准株获得了较群体感应基因LuxS的突变株更多的细胞生长。在BHI中标准株和突变株均能够形成生物被膜,但结构存在差异：标准株形成光滑且均匀分布的生物被膜,突变株的生物被膜形态粗糙。于扫描电镜下观,与生长在补充了2%葡萄糖的TPY液体培养基中的变链菌标准株相比,LuxS基因突变株所形成的生物被膜表现型未见明显差异。在补充了2%蔗糖的TPY液体培养基中生长时,LuxS基因突变株所形成的生物被膜的表现型在形态学上与标准株形成的生物被膜有明显的不同,LuxS基因突变株形成较大的团簇状菌落,生成生物被膜的能力下降。结构相对粗糙,呈松散的蜂房状,生物被膜基质间有较大的间隙,而标准株生物被膜呈相对融合的外观,分布更加均衡。当用标准株上清液来弥补突变株时,形成的聚集物要小的多,此时形成的生物被膜结构介于标准株和突变株之间。结论：本研究通过常规生化鉴定、革兰氏染色涂片鉴定和生长曲线测定等方法证实了变链菌标准株与LuxS突变株在生长特性上有一定的差异性,LuxS基因突变可以抑制变链菌的生长,并阐述了两菌株在生物学性状方面的异同。此外,通过建立LuxS基因缺失突变株的生物被膜体外模型,用结晶紫染色等方法观察了变链菌生物被膜结构,证实了LuxS基因突变对其形成生物被膜的能力有一定影响,扫描电镜下分析,两种菌株均表现出形成生物被膜的能力,但LuxS基因突变株形成生物被膜的能力有所减弱,结构发生改变,标准株能弥补LuxS基因突变株的生物被膜的变化表型,依赖于LuxS的群体感应系统会影响变链菌生物被膜的形成。
[Abstract]:Objective: the purpose of this study is through the use of conventional biochemical identification, gram elaborated between Streptococcus mutans standard strain strain in biological characters and LuxS mutation stain identification and determination of growth curve and other methods. In addition, the establishment of the LuxS gene deletion mutant biofilm model in vitro by crystal violet staining, variable chain the bacterial biofilm structure, mutation of LuxS gene on the biological effects of membrane formation by ability, and with the aid of scanning electron microscopy LuxS mutant strain and standard strain in the biofilm, and lay the foundation for the further study of the regulatory mechanism of LuxS signaling system on the cariogenicity of virulence.
Methods: the chain will become standard strain and LuxS mutant strain were cultured in culture medium for colony in contain erycin or not TSA solid, to observe the growth of two colonies and conventional biochemical test, Gram staining to compare the growth characteristics of two kinds of strains. The recovery of 24h standard strain and LuxS mutant on line ultraviolet spectrophotometer at 600nm prepared absorbance A=1.0 bacterial suspension. The suspension will reserve 1000 times dilution inoculated in BHI liquid medium, a constant temperature of 37 DEG C shaker in the incubation time sampling measurements of A were observed to compare the differences in the growth curve of the LuxS gene. Mutant strains and standard strains inoculated in BHI liquid medium, after 48h, remove the corresponding microplate, crystal violet staining of biological membrane, observe the morphological changes of bacterial biofilm. Early prepared sterile enamel slabs placed 6 Hole Fine Bao Pei A board, and numbered the 1-6 holes, each hole placed 4 tablets were taken S.mutan standard strain and LuxS mutant of A600=1.0 bacterial suspension inoculation to each hole 1ml (including 1,4 liquid fungus inoculation standard strain 2,3,5,6 four hole inoculated LuxS mutant). Uniform drop to the enamel surface of the disc, standing about 2min after adding 5mlTPY into each hole liquid medium (the 1,2,3 is TPY solution containing 2% 4,5,6 glucose is TPY solution containing 2% sucrose), and then advance the prepared standard strains of 500 mu l of the supernatant was added to 3,6 two holes. The bacteria liquid at 37 DEG C under anaerobic conditions in cultured 24h, scanning electron microscope in the formation of enamel slabs on the sterile biofilm, and overall evaluation to a comparison of two strains of biofilm formation ability.
Results: in TSA-Eymr solid medium containing erythromycin, basic standard strains can grow, and the growth status of the normal mutation strains. In the medium containing erythromycin TSA solid, there was no difference in colony morphology of two strains, microscopically standard strain was long chains and tangled, and the mutant bacteria in short chains, the formation of long chain less. By means of variable chain standard strain and LuxS mutant strain growth curves were found both in growth mode has no significant difference, but in stable growth, there are some differences in bacterial saturation are obtained with two standard strains the quorum sensing gene LuxS mutant growth. More cells in BHI standard strain and mutants were able to form biofilms, but there are differences in the structure: the standard strain to form a smooth and uniform distribution of biofilm process Strain biofilm morphology by scanning electron microscope. The rough concept, and growth in added 2% glucose TPY liquid culture medium of Streptococcus mutans standard strains compared with LuxS mutant in the biofilm phenotype. There is no significant difference in TPY added 2% sucrose liquid medium. When the LuxS gene mutation phenotype strains in the biofilm formation and standard strains in morphology of biofilm were significantly different, LuxS mutant formed clusters like the larger colonies, generating creature falling film ability. The structure is relatively rough, loose honeycomb, creature there is a large gap between the membrane matrix, and the standard strains of biofilm showed relatively fusion appearance, distribution is more balanced. When using standard strains of supernatant to compensate for the mutant, the formation of aggregates is much smaller, the formation of biofilm structure between standard strain and Among the mutant strains.
Conclusion: This study by conventional biochemical identification, Gram staining identification and determination of growth curve and other methods confirmed the Streptococcus mutans standard strain and LuxS mutant on growth characteristics have some differences, the mutation of LuxS gene can inhibit the growth of S.mutans, and expounds the similarities and differences between the two strains in biological characters. In addition, through the establishment of LuxS gene deletion mutant biofilm model in vitro, with the crystal violet staining method to observe the S.mutans biofilm structure, confirmed that LuxS gene mutation had certain influence on its ability to form biofilm, scanning electron microscope analysis, two strains showed the formation ability of creatures the film, but the LuxS mutant with the ability to form biofilm weakened, change the structure and standard strains can make up for the phenotypic change of LuxS gene mutation strains of biofilm, quorum sensing system relies on LuxS The association affects the formation of the biofilm of the Streptococcus mutans.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【参考文献】