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.     

伴放线放线杆菌形态变化对菌体表面疏水性影响的研究

目的研究伴放线放线杆菌形态变化对菌体表面疏水性的影响。方法采用碳氢化合物法检测伴放线放线杆菌粗糙型和光滑型的菌体表面疏水性,观察同一菌株不同表型疏水性的变化。结果伴放线放线杆菌粗糙型和光滑型菌体表面具有疏水性。14株粗糙型伴放线放线杆菌菌体表面疏水率高于4株光滑型,差异有统计学意义（P〈0．05）。4株同源的粗糙型与其光滑型转变株比较得出除1株外,其余3株菌两种表型的菌体表面疏水率差异无统计学意义（P〉0．05）。结论伴放线放线杆菌形态变化可引起菌体表面疏水性的改变,粗糙型转变为光滑型后菌体表面疏水性减弱。     

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。     

伴放线放线杆菌血清型分析

目的 PCR法检测伴放线放线杆菌（Actinobacillus actinomy＇etemcomitans，Aa）临床分离菌株血清型，分析其与flp-1基因型的关系。方法用血清型特异性引物，通过普通PCR和多重PCR的方法对60株Aa临床分离菌株的血清型进行鉴定，并分析其与flp-1基因型的关系。结果 60株Aa临床分离菌株中血清型c型63，33％，e型23．33％，b型6.67％，a，f型各占3．33％，未检测到d型菌株；在24名被检测者中，15名检测到c型An菌株，3名检测到b型菌株，各有2名分别检测到a、e、f型菌株。fip-1基因型Ⅰ型菌株的血清型均为a型，40株Ⅱ型菌株中38株为c型，Ⅳ型菌株均为b型，11株Ⅴ型菌株中9株为e型，Ⅵ型菌株均为e型。结论 Aa血清型分布以c型为主，fip-1基因型与菌株血清型存在一定对应关系。     

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。     

伴放线共生放线杆菌表面相关物质的骨吸收活性研究

目的：通过对伴放线共生放线杆菌（Aa)表面相关物质（SAM）的提取，纯化，研究其潜在的骨吸收活性，以揭示SAM在牙周病变过程中的作用。方法：Aa国际标准菌株培养，SAM提取，过Sepharyl-S200层析柱和DEAE－Sephacel层析柱，所有的管用酚硫法测其碳水化合物含量，纯化后鉴定其骨吸收活性.结果：冻干的Aa(1.0g)产生0．1375g粗提物，通过Sepharyl-S200时出现单峰，通过DEAE－Sephacel出现两个峰，骨吸收实验证明SAM有骨吸收活性。结论：Aa的SAM在骨吸收方面具有极大潜能，提示SAM在牙周病骨损害上起到重要作用。     

白屈菜红碱对伴放线放线杆菌的抑制作用研究

中草药白屈菜(Chelidonium majus L.)提取物白屈菜红碱对致龋菌的生长具有较强的抑制作用,但其对牙周致病菌是否有作用,未见报道。本实验对白屈菜红碱对伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)的抑制作用进行了初步研究,报告如下。1材料与方法1.1菌液制备将复苏48h的Aa ATCC29523接种于BHI液体培养基中,37℃厌氧培养24h,离心后收集细菌,用无菌生理盐水调在540nm处吸光度为1.0的菌悬液。1.2纸片制备选中性定性滤纸,用打孔器打成直径6mm的圆片,干热灭菌。在无菌操作下加不同浓度的药液1mL,4℃浸泡过夜,干燥机抽干,4℃…     

口腔链球菌对伴放线嗜血菌生长影响的体外实验

目的：研究变异链球菌、血链球菌、远缘链球菌、唾液链球菌对伴放线嗜血菌生长的影响。方法：制备伴放线嗜血菌BHI琼脂板，将4种口腔链球菌菌悬液滴注于其上，兼性厌氧培养48h，观察有无抑菌现象。分别将4种口腔链球菌与伴放线嗜血菌等体积等浓度混合制成双菌种悬液在液体BHI中培养，每隔4h取浮游菌悬液梯度稀释，接种于BHI琼脂板上培养48h，计数伴放线嗜血菌占菌落总数的百分比：扫描电镜观察双菌种生物膜生长情况。结果：琼脂扩散实验显示，变异链球菌、血链球菌、远缘链球菌周围出现抑菌圈，唾液链球菌周围未出现抑菌圈。伴放线嗜血菌所占双菌种混合细菌的比例随时间延长呈下降趋势（p〈0．05）。生物膜观察显示，单菌种伴放线嗜血菌形成生物膜的时间较口腔链球菌时间长；双菌种混合培养时伴放线嗜血菌的量随时间推移而减少。结论：4种口腔链球菌抑制伴放线嗜血菌生长。     

放线共生放线杆菌表面相关物质对人牙龈成纤维细胞的增殖抑制作用

目的：观察放线共生放线杆菌表面相关物质对人牙龈成纤维细胞的增殖抑制作用。方法：采用细胞计数法和图像分析法观察对人牙龈成纤维细胞的生长抑制作用。结果：此物质抑制作用明显，能抑制细胞的分裂、增殖，使细胞、细胞核面积增大，１００ｍｇ／Ｌ对与对照组差别显著（Ｐ＜０．０５），且细胞无死亡现象。结论：此物质可明显抑制人牙龈成纤维细胞的生长、增殖，在牙周病病理变化过程中起重要作用     

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.     

Osteoblast-like cells are sensitive to submicron-scale surface structure

OBJECTIVES: Studies showing that osteoblasts exhibit a more differentiated phenotype on rough titanium (Ti) surfaces and osteoclast-resorbed bone surfaces used materials characterized by average peak to valley distance (Ra). Other surface features impacting the cells include distance between peaks, curvature of the valleys, and relative distribution of flat and smooth regions. We used novel Ti surfaces prepared by electrochemical micromachining as models to examine specific contributions of individual design features to osteoblast response. Results show that micron-scale topography modulates cell number, cell morphology and prostaglandin E2 (PGE2). In the presence of the appropriate microtopography, submicron-scale rugosity modulates differentiation and transforming growth factor-beta1 (TGF-beta1) levels. In this study, we examined the role of different types of submicron-scale structures. MATERIAL AND METHODS: Thirty micrometer diameter craters on Ti disks were produced by photolithography resulting in an electropolished smooth surface, and arranged so that inside crater area vs. outside flat area was 6 (30/6). Submicron-scale structures were superposed by acid etching and porous anodization. Ra's were 700, 400, 60 nm on acid-etched, porous anodized and smooth 30/6 surfaces, respectively. RESULTS: MG63 osteoblast-like cells were sensitive to submicron-scale architecture. Cell morphology on anodized surfaces was similar to morphology on smooth surfaces, whereas on etched surfaces, cells had a more elongated differentiated shape. Cell number was greatest on smooth surfaces > anodized > etched. Osteocalcin and PGE2 were affected in a reverse manner. Active TGF-beta1 was greatest on etched 30/6 surfaces > anodized > smooth; latent TGF-beta1 was elevated on all rough surfaces. CONCLUSIONS: These results support our previous observations that submicron-scale structures modulate osteoblastic phenotype and show that the physical properties of the submicron-scale structures are important variables in determining osteoblast response to substrate topography.     

LuxS基因缺失对变异链球菌生物学性状的影响

目的:观察LuxS基因突变对变异链球菌生物性状的影响。方法:将变链菌标准株和LuxS突变株分别培养于TSA、TSA-Cmr、BHI培养基培养48 h,通过菌落形态观察、常规生化检测、革兰染色涂片观察和生长曲线,比较两种菌株的生长特性;体外建立LuxS基因突变株和标准株的生物被膜模型,结晶紫染色,观察比较两菌株形成生物被膜的能力。结果:在含氯霉素的TSA-Cmr固体培养基中增塑,标准株基本不能生长,而突变株的生长状况基本正常。在不含氯霉素的TSA固体培养基中培养,两种菌株的菌落形态没有明显差别,革兰染色镜下观察,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。生长曲线观察发现:两菌株在生长模式上基本一致,均呈典型的"S"型曲线,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,即11.5～22.5 h之间各时间点标准株的A值均明显高于LuxS突变株(P<0.05)。在BHI培养基中两菌株均能形成生物被膜,但结构存在差异:标准株形成光滑且均匀分布的生物被膜,而突变株的生物被膜形态较粗糙。结论:LuxS基因突变对变异链球菌的生长以及生物被膜形成能力等生物学性状有一定影响。     

Roles of Streptococcus mutans dextranase anchored to the cell wall by sortase

In order to clarify the role that sortase (SrtA) plays in anchoring dextranase (Dex) to the cell wall of Streptococcus mutans, both Dex- and SrtA- mutants were constructed by insertional inactivation of the respective genes. Western blot analysis with a Dex antiserum showed that in the srtA mutant the Dex was not bound to the cell wall but was secreted into the culture supernatant. In contrast, in the wild type, Dex remained cell-wall-associated. Biological properties of the srtA mutant were examined in dextran fermentation, colony morphology and adherence to a smooth surface. The srtA mutant, as well as the wild type, retained the ability to ferment dextran. However, the colony morphology of the srtA mutant on Todd Hewitt agar containing sucrose was much larger than that of the wild type and showed a ring-like structure. In addition, the srtA mutant was more adhesive to a smooth surface than the wild type when sucrose was present. However, the adhesion of the srtA mutant remarkably decreased by addition of exogenous dextranase. These studies suggest that the SrtA mediates Dex-anchoring to the cell wall in S. mutans, and cell wall-anchored Dex plays a role in controlling both the adhesive properties of extracellular glucan and the ability to utilize extracellular glucan as a nutrient source. In contrast, extracellular Dex is only responsible for degrading extracellular glucan as a nutrient source.     

Effects of opaque and porcelain surface texture on the color of ceramometal restorations

A total of 80 ceramometal samples were constructed with two different types of surface texture in the opaque layer (glossy and dull). Porcelain was applied to these samples with two different surface textures (smooth and rough). Samples were made in 2 mm thick porcelain shades B1 and A3. In addition, 10 1 mm samples were constructed with shade A3 having dull opaque, smooth, and rough porcelain surface textures. Ten samples of opaque only were constructed with two different surface textures (glossy and dull).Each sample was analyzed in the recording spectrophotometer, and CIE color notations were calculated. These data were translated to the Munsell Scales (Hue, Chroma, and Value). Two randomly selected samples of each group were then reevaluated in the spectrophotometer using a device which excluded the specular reflectance factor.The three dimensions of color—Hue, Chroma, and Value—were statistically analyzed and compared by the Student-Newman Keuls procedure. Each set was submitted to a correlation test to determine significant differences.     

Transformation of Streptococcus sanguis to a rough colonial morphology with an increased ability to adhere.

DNA isolated from a rough variant of Streptococcus sanguis transformed smooth, competent strains of Strep. sanguis into variants with rough colonial morphology. The rough variant showed increased adherence ability and this property was transferred. A similar rough colonial morphology of Strep. mutans could not be transferred to these competent Strep. sanguis strains by transformation, suggesting that the rough morphology of Strep. mutans and that of Strep. sanguis are determined by different genetic markers. The smooth and rough variants of Strep sanguis had different growth rates. The logarithmic phase of the smooth wild-type was significantly shorter than that of the rough variant.     

Optimization of surface micromorphology for enhanced osteoblast responses in vitro.

In vitro cellular responses of osteoblast-like cells were studied on titanium surfaces with different surface morphologies. Surface profilometry was used to determine whether rough or smooth surfaces with regular or irregular morphologies can be produced by conventional fabrication techniques. Significantly higher levels of cellular attachment were found using rough, sandblasted surfaces with irregular morphologies. These results correlate with recent in vivo findings and suggest that implants should be prepared with roughened surfaces at bony contact areas.     

Phenotypes, serotypes and antibiotic susceptibility of Swedish Porphyromonas gingivalis isolates from periodontitis and periodontal abscesses

This study was conducted to reveal phenotypic, serological subtypes and antibiotic susceptibility among fresh isolates of Porphyromonas gingivalis in a Swedish population with periodontitis and periodontal abscess. Fifty-five subgingival strains were isolated and tentatively designated as P. gingivalis from 55 consecutive paper-point samples taken from 51 patients with periodontitis (at least one site with >6-mm pocket depth) in Sweden and were sent in for microbiological evaluation. Eight P. gingivalis strains from periodontal abscesses were also included. Four P. gingivalis strains served as reference and another four type strains were included. The strains were characterized by colony morphology, biochemical tests, enzyme profile, gas-liquid chromatography and antibiotic susceptibility. The strains were further characterized for whole cell protein profiles using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were identified to serotype by specific monoclonal antibodies. Among the 55 P. gingivalis strains 35 had smooth (S), 13 rough (R) and seven semi-rough colony morphologies. All strains were phenotypically homogeneous in biochemical tests, enzyme profile and antibiotic susceptibility. All strains produced phenylacetic acid and alpha-fucosidase. Almost all (96%) of the subgingival strains, but relatively fewer (62%) of the abscess strains, belonged to serotype A. Two subgingival and three abscess strains were classified as serotype B. No specific SDS-PAGE protein profiles were recorded for the two serotypes. The P. gingivalis strains from Swedish periodontitis cases showed homogeneity in terms of biochemical phenotypes and antibiotic susceptibility patterns. The strains fell into two serotypes, of which serotype A predominated in the periodontitis cases and serotype B was overrepresented in periodontal abscesses.