临床变异链球菌及其412c基因缺陷菌株的功能初步分析

Preliminary analysis in functions of clinical Streptococcus mutans and 412c-deficient mutant strains

目的分离、纯化和鉴定临床变异链球菌株,并构建该临床菌株的412c基因缺陷菌株,比较两者生长速率和致龋性。方法随机收集无龋健康成人口腔牙菌斑样本,在唾液/变异链球菌杆菌肽琼脂(MSA)培养基中进行厌氧培养和筛选,通过菌斑形态观察、革兰氏染色、细菌生化反应和聚合酶链式反应(PCR)等方法对获得的临床分离株进行鉴定;将线性化重组质粒转入临床变异链球菌中进行同源重组,获得412c(hit)基因缺陷菌株;在不同pH值脑心浸液(BHI)培养基中,比较两者的生长速率和耐酸特性;在含3%蔗糖的BHI培养基中,比较两者的产酸能力。结果无龋健康成人口腔牙釉质表面菌斑在MSA培养基中生长的菌落形态均为深蓝色半球颗粒状突起、表面粗糙、边缘不齐;革兰氏染色后,显微镜油镜下可见革兰阳性蓝紫色球状菌体,呈长链状和短链状分散;生化反应和412c基因片段PCRSanger测序鉴定结果显示,临床变异链球菌为“等效变异链球菌UA159”菌株。将hit-UP-pFW5-Down重组质粒线性化后转入临床变异链球菌中进行同源重组并筛选出412c基因缺陷变异链球菌株,该缺陷菌株生长速率相较其亲本菌株异常快速(与模式菌株ATCC 25175的缺陷菌株相似),其产酸能力略弱于亲本菌株,耐酸性则显著强于亲本菌株。结论临床分离变异链球菌株为“等效变异链球菌UA159”菌株,其412c基因与细菌生长调控相关,而412c基因缺陷变异链球菌株的生长速率显著高于其亲本菌株,更具致龋潜力。

Objective To isolate,purify and identify clinical Streptococcus mutans(S.mutans)strains,construct theiR412c-deficient mutant strains,and investigate on theiRproliferation and cariogenicity.Methods Dental plaque samples were randomly collected from healthy adults without caries,were anaerobically cultivated on saliva oRS.mutans bacitracin AGAR(MSA)medium and anaerobion were screened out.The isolated S.mutans strains were identified by colony morphology,Gram staining,biochemical test and polymerase chain reaction(PCR).The linearized recombinant plasmids were transformed into clinical S.mutans strains foRhomologous recombination to obtain 412c(hit)gene defect strains.The growth rate and acid resistance in the brain heart immersion(BHI)medium with different pH values.In BHI medium containing 3%sucrose,the acid production capacity of the two kinds of strains was compared.Results The morphology of the dental enamel surface plaque of healthy adults without caries growing in MSA medium was dark blue hemispheric granulaRprotrusions with rough surface and uneven edges.Using Gram staining,there were many long and short chained coccus-shaped bluish violet bacterium undeRan oil-microscopy.The isolated S.mutans was identified as"an equivalent S.mutans UA159"by biochemical test and PCR-SangeRsequencing of 412c gene segment.The hit-UP-pFW5-Down recombinant plasmids were transformed into the genetic competence of the isolated S.mutans strains.The 412c-deficient mutant strains were screened out.SimilaRto S.mutans ATCC 25175,the growth rate of the 412c-deficient mutant strains increased greatly compared with theiRparental S.mutans strains.Conclusion The clinical isolate of S.mutans was"equivalent S.mutans UA159"strain,in which the 412c gene is growth-phase regulated,412c-deficient S.mutans strains have significantly higheRgrowth rates than its parental strain and have more cariogenic potential.