放线共生放线杆菌显微形态学的研究

目的 观察放线共生放线杆菌不同菌落菌株形态特点，探讨其致病的形态学基础。方法 粗糙型和光滑型两种菌落形态的细菌固体培养2天-4天应用电子显微镜阴性染色的方法比较观察放线共生放线杆菌菌体表面结构特点。结果 粗糙型菌落菌体菌毛丰富；刚刚转变后的光滑型菌落菌体仍可存在菌毛，但这种菌落继续传代菌毛可完全失去，菌毛表现为几种形态，细密或稀疏，也可成束状或网状，粗糙型菌体染色深，不均匀，菌体边缘不规则，视野不干净，多次传代后的光滑型菌落，菌体染色浅且均匀，菌体饱满透明，视野干净，粗糙型可以见到膜泡样的结构，但量很少。结论 粗糙型和光滑型菌的菌体形态存在明显的差异。主要表现为菌毛。     

伴放线放线杆菌形态变化对白细胞毒素分泌影响的研究

目的观察伴放线放线杆菌形态变化对白细胞毒素分泌的影响。方法选择粗糙型和光滑型伴放线放线杆菌各8株,应用聚丙烯酰胺凝胶电泳,检测液体培养12、24、48、60、72h的菌体及培养上清液中116kDa大小白细胞毒素蛋白条带的情况,应用超滤法分离纯化培养上清液蛋白,应用台盼蓝染色排除法检测上清液蛋白白细胞毒素活性。结果粗糙型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳均可见116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带均出现于培养24和48h;光滑型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳结果均缺少116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带出现于培养12和24h;实验菌株培养上清液提取蛋白均具有白细胞毒素活性。结论伴放线放线杆菌粗糙型和光滑型菌株均可分泌具有直接杀灭人多形核白细胞活性的白细胞毒素,但粗糙型菌株分泌白细胞毒素的时间晚于光滑型。     

伴放线放线杆菌光滑株与粗糙株生长及磷酸胆碱表达的比较研究

目的 绘制伴放线放线杆菌同一菌株不同表型的生长曲线,测定菌体蛋白表达上的差异。方法 采用液体培养基TSB培养伴放线放线杆菌分离株D7S及其光滑型菌株D7SS,紫外分光光度计检测细菌密度,连续观测30h,绘制生长曲线。聚丙烯酰胺凝胶(SDS-PAGE)电泳后考马斯亮蓝染色比较两种表型的细菌蛋白的差异。免疫印迹法(Western Blot)观察两种表型细菌蛋白对单克隆抗体TEPC-15的反应情况。结果 D7SS菌株比D7S菌株更早进入对数期和衰亡期;两型细菌全细胞蛋白电泳考马斯亮蓝染色时蛋白带未见明显差异;免疫印迹分析D7S菌株在分子质量接近10ku的蛋白中存在磷酸胆碱,而D7SS中未发现。结论 伴放线放线杆菌D7S和其光滑型菌株D7SS的生长曲线以及对TEPC-15抗体的反应性明显不同。     

伴放线放线杆菌粘附特性研究

目的分析伴放线放线杆菌的粘附特性及菌毛结构基因tip-1的遗传多样性对菌株粘附活动的影响。方法检测不同孵育条件下5种tip-1基因型临床分离菌株和光滑型菌株的粘附活动。结果临床分离菌株的粘附量随菌液浓度,孵育时间的增加而增加。tip-1基因型Ⅱ型菌株的粘附量高于其它4型菌株,光滑型菌株的粘附量低于临床分离菌株。生理温度下菌株粘附数高,低温下明显降低。厌氧条件和有氧条件下的粘附量无显著性差异。结论伴放线放线杆菌临床分离菌株的粘附存在时间和菌量依赖性,并要求一定新陈代谢活性,粘附效率在氧浓度改变时没有明显变化。伴放线放线杆菌表型影响菌株的粘附作用。不同tip-1基因型菌株粘附能力存在差异,Ⅱ型菌株粘附能力最强。     

伴放线放线杆菌flp-1基因遗传多样性分析

目的:分析伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)临床分离菌株菌毛结构基因flp-1的遗传多样性和flp-1基因与菌株表型之间的关系.方法:对从不同牙周状况患者口腔中分离的60株Aa(57株粗糙型,3株传代转变成光滑型)和6株Aa标准菌株(光滑型)进行flp-1基因扩增,并通过PCR限制性片段长度多态性(PCR-RFLP)法分析临床分离菌株flp-1基因的遗传多样性.结果:所有57株粗糙型菌株和9株光滑型菌株都检测到flp-1基因;60株临床分离菌株检测到5种flp-1基因型,其中基因型2型占67%,共有40株.结论:Aa菌株表型的改变不伴有flp-1基因缺失;Aa临床分离菌株flp-1基因具有遗传多样性,基因型主要为2型.     

伴放线放线杆菌菌落生长形态变化的观察

目的：观察伴放线放线杆菌（Actinobacillus actinomycetemcomitans,Aa)从粗糙型到光滑型的转变过程，认别Aa在实验室传代过程中出现的不同生长形态。方法：从牙周炎患者龈下菌班中分离出的原代菌株8株，应用固体及液体培养基连续传代，液体培养每次传代的同时接种固体培养基观察相应的菌落形态。结果：液体培养获得3株光滑型转变株。菌落的变化从粘附的小菌落到沉淀的大菌落到完全的均匀生长，转化过程大约需要7－8代。在这一过程中相应传至固体培养基上生长的Aa从粘附的半透明的小菌落变大、不透明并失去粘附的特性，又随着边缘的扩散变为扁平，透明度也增加；与此同进内部的星形结构逐渐变简单、变小，最后消失。固体培养未获得典型的转变株。结论：Aa从粗糙型到光滑型的转变是一个菌落湿度逐渐增加，体积逐渐增大，并逐渐失去内部结构的过程。这一过程至少可以看到半透明突起的粗糙型，不透明突起的光滑型和近乎透明的扁平光滑型3种菌落形态。     

伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的体外研究

目的研究伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的作用。方法选取10名全身及牙周组织健康受试者,分离外周血淋巴细胞,在有／无伴放线放线杆菌情况下培养0—96h,用荧光探针（AnnexinV—FITC、PI、CD69-TC7）进行标记,并进行流式细胞仪检测。结果全淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为13．42±2．88、22．74±2．18、46．92±4．28,全淋巴细胞组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为8．46±2．53、6．36±2．36、9．36±2．67,2组间存在明显差异（P〈0．01）。CD69加淋巴细胞加伴放线放线杆菌组和CD69＋淋巴细胞组AnnexinV＋／PI-细胞百分数除48h外的4个时间点上都无明显差异（P〉0．05）。CD69＋淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在各个时间点上都明显高于全淋巴细胞加伴放线放线杆菌组（P〈0．01）。结论伴放线放线杆菌能够诱导人外周血淋巴细胞活化,并且能够通过活化促进其凋亡。     

口腔微生物与免疫学

白色念珠菌在义齿软衬表面滋生及检测,伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的体外研究,伴放线放线杆菌形态变化对菌体表面疏水性影响的研究,大蒜素对变形链球菌抑制作用的体外研究,五倍子、白芨、龙胆草对变形链球菌影响的研究.     

伴放线放线杆菌菌毛研究进展

伴放线放线杆菌是侵袭性牙周炎的可疑致病菌,菌毛是其重要的致病因子。本文对伴放线放线杆菌菌毛的形态、相关基因和蛋白、基因表达的相关调控、致病作用以及免疫原性进行了综述。     

伴放线放线杆菌菌毛研究进展

伴放线放线杆菌是侵袭性牙周炎的可疑致病菌，菌毛是其重要的致病因子。本文对伴放线放线杆菌菌毛的形态、相关基因和蛋白、基因表达的相关调控、致病作用以及免疫原性进行了综述。     

Cell surface hydrophobicity and electrokinetic potential of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus

Laboratory strains and fresh isolates of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus were examined for cell surface hydrophobicity and for electrokinetic properties under different experimental conditions. Fresh isolates of A. actinomycetemcomitans and H. aphrophilus were hydrophobic. Laboratory strains of A. actinomycetemcomitans were 20-30% less hydrophobic than fresh isolates. No difference was observed between laboratory and fresh isolates of H. aphrophilus. The pH of the suspending buffer, growth phase or incubation atmosphere did not significantly affect the hydrophobicity of the 2 species, whereas agar cultures of H. aphrophilus were less hydrophobic than broth cultures. All A. actinomycetemcomitans strains treated with sterile filtered saliva showed a concentration-dependent decrease in hydrophobicity of at most 30%. H. aphrophilus strains were not affected by the same treatment. Laboratory strains of H. aphrophilus were more negatively charged than A. actinomycetemcomitans, whereas fresh isolates of the 2 species exhibited similar surface charge. In the presence of saliva the mean cell surface charge of laboratory strains decreased by 56% for A. actinomycetemcomitans and by 73% for H. aphrophilus. The results indicate that the 2 species differ in expression of cell structures accounting for hydrophobicity and surface charge and that environmental factors might differently influence the physical properties of the two species analyzed.     

Environmental influences on Actinobacillus actinomycetemcomitans biofilm formation

Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans have an adherent, rough colony morphology that transforms into a minimally adherent, smooth colony phenotype during successive in vitro passage. The objectives of this study were: (1) to compare biofilm formation of the rough (RVs) and smooth variants (SVs) of several strains of A. actinomycetemcomitans grown under various environmental conditions and (2) to examine the dynamics of biofilm formation. A microtitre plate biofilm assay was used to evaluate biofilm formation of strains grown in broth with modified salt concentration and pH, and to evaluate the effect of pre-conditioning films. Scanning electron microscopy (SEM) was used to monitor microscopic changes in morphology. Dynamics of biofilm formation were measured in a flowcell monitored by confocal microscopy. The RVs generally produced greater biofilm than the SVs. However, medium-dependent differences in biofilm formation were evident for some rough/smooth pairs. The RVs were more tolerant to changes in salt and pH, and more resistant to chlorhexidine than the SVs. Horse serum virtually eliminated, and saliva significantly reduced, biofilm formation by the SVs in contrast to the RVs. SEM revealed no alteration in morphology with change of environment. In a flowcell, the RVs produced towers of microcolonies anchored by a small contact area, whereas the SVs produced an open architecture of reduced height. After 7 days in a flowcell, the rough to smooth phenotype transition could be demonstrated. In conclusion, strain, growth medium and conditioning film all affect biofilm formation. The RVs produce biofilms of unique architecture that may serve to protect the bacterium from environmental perturbations.     

Probe-specific DNA fingerprinting applied to the epidemiology of localized juvenile periodontitis

Although Actinobacillus actinomycetemcomitans has been recognized as a primary etiological agent in localized juvenile periodontitis, questions remain concerning the source of infection, mode of transmission, and relative virulence of strains. DNA fingerprinting analysis, using a randomly cloned chromosomal DNA fragment as a probe, revealed that previously characterized strains of A. actinomycetemcomitans displayed significant restriction site heterogeneity which could be applied to the typing of clinical isolates of this bacterium such that individual strains or variants could be traced within subjects from localized juvenile periodontitis families. Hybridization data derived from an analysis of bacterial isolates obtained from families participating in an ongoing longitudinal study of the disease showed that a single individual could be infected with more than one strain or variant of A. actinomycetemcomitans and that various members of the same family could harbor different strains or variants of the bacterium. In several cases the clinical isolates were matched to characterized laboratory strains by comparing hybridization patterns generated by digestion of the DNA with several restriction enzymes in independent reactions. Thus, probe-specific DNA fingerprinting of A. actinomycetemcomitans will permit us to determine if particular strains or variants are frequently associated with sites of periodontal destruction. Attention could then be focused on determining the virulence properties of those strains or variants that have in vivo significance.     

采用PCR方法鉴别伴放线放线杆菌的6种血清型

目的：探索采用PCR的方法对伴放线放线杆菌的不同菌株进行血清型分类。方法：根据伴放线放线杆菌不同血清型特异性多糖抗原基因序列设计6对不同的寡核苷酸引物，用这6对引物分别对所选择伴放线放线杆菌6种不同的血清型菌株各3株，共18株，其中参考菌株6株，系ATCC29523（血清型a），ATCC43718（血清型b），ATCC33384（血清型c），IDH781（血清型d），IDH1705（血清型e）以及CU1000（血清型f），其余12个菌株均为临床分离株的DNA进行PCR扩增分析。结果：每一对引物均针对相应的血清型产生特异性单一条带PCR产物，产物大小分别为428bp（a），298bp（b），559bp（c），690bp（d），211bp（e），232bp（f）。全部18个菌株均能够被准确识别，无交叉反应。结论：PCR方法可以快速准确地鉴别伴放线放线杆菌目前已知的全部6种血清型。     

Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans

Actinobacillus actinomyetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzas, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans , in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.     

Phenotypic stability and plasmid detection in Actinobacillus actinomycetemcomitans.

The stability of hemolytic activity, antibiotic resistance and plasmid detection in Actinobacillus actinomycetemcomitans isolates were studied. These characteristics were stable for all experimental conditions. All tested isolates lost or changed some phenotypic characteristics such as colonial morphology and growth in liquid medium.     

Prevalence of Actinobacillus actinomycetemcomitans serotypes in Japanese patients with periodontitis

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against. A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP-PCR) to fingerprint clinical isolates of different serotypes. The AP-PCR genotype among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetecomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogenous in the periodontally diseased sites of patients with periodontitis.