伴放线放线杆菌与致龋菌生长关系的体外研究

目的:观察伴放线放线杆菌(A.actinomycetemcomitans)与4种致龋菌,即变异链球菌(S.mutans)、嗜酸乳杆菌(L.acidophilus)、内氏放线菌(A.naeslundii)、粘性放线菌(A.viscosus)在体外共培养时相互间的生长抑制作用.方法:改良琼脂扩散法和双菌种液体培养观察A.actinomycetemcomitans与S.mutans、Lacidophilus、A.,naeslundii、A.viscosus生长的相互关系;扫描电镜及菌落计数观察双菌种生物膜形态和细菌比例的差异.实验数据用SPSS10.0软件包进行两样本均数的t检验.结果:琼脂扩散法抑菌实验表明,A.actinomycetemcomitans对S.mutans、L.acidophilus、A.naeslundii、A.viscosus的生长无抑制作用,S.mutans、L.acidophilus、A.naeslundii、A.viscosus可抑制,A.actinomycetemcomitans的生长.在双菌种液体培养中,A.actinomycetemcomitans所占比例随时间逐渐下降,差异具有显著性(P＜0.05):A.actinomycetemcomitans培养12h后,所占比例呈下降趋势,24h在S.mutans和L.acidophilus组中的比例下降为0.生物膜的观察表明,单菌种A.actinomycetemcomitans成块状散在黏附,无完整生物膜形成,4种致龋菌在48h均形成结构完整的生物膜,具有三维立体结构.与单菌种相比,加入A.actinomycetemcomitans后,致龋菌的生物膜形态未发生明显改变.更换培养液后,A.actinomycetemcomitans在混合菌种生物膜中的比例仍随时间延长下降,72h降低到最低点,各时间点的差异具有显著性(P＜0.05).结论:体外培养4种致龋菌可抑制A.actirtomycetemcomitans的生长.     

噻唑蓝比色法检测变异链球菌、血链球菌、伴放线菌嗜血菌的研究

目的探讨噻唑蓝（MTT）法用于口腔常见细菌（变异链球菌、血链球菌、伴放线菌嗜血菌）活菌计数的可行性及具体应用过程中的实验条件。方法本实验以菌落形成单位（CFU）法为标准对照。实验通过改变比色的波长、反应时间、试剂剂量、细菌菌龄等实验条件,获得MTT法测量常见口腔细菌的一系列参数,对变异链球菌、血链球菌、伴放线菌嗜血菌进行活菌计数,并与CFU法所测的细菌数量相比较。结果MTT法在测量变异链球菌时,最佳的检测波长为510 nm,最佳的测定细菌范围是1.5×105～1.0×107 CFU·mL-1。MTT法在测量血链球菌时,最佳的检测波长为545 nm,最佳的测定细菌范围是1.5×105～2.0×107 CFU·mL-1。MTT法在测量伴放线菌嗜血菌时,最佳的检测波长为557 nm,最佳的测定细菌范围是1.0×106～5.0×107 CFU·mL-1。MTT法与菌落形成单位法的测量结果是一致的,并且对于不同菌龄的变异链球菌、血链球菌、伴放线菌嗜血菌均适用。结论MTT法可以用于检测变异链球菌、血链球菌、伴放线菌嗜血菌等口腔常见细菌的活菌数量,并且具有快速、方便等优点。     

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。     

抗伴放线放线杆菌IgY的制备及抑菌试验研究

目的:观察伴放线放线杆菌诱导母鸡产生特异性IgY抗体情况,以及其抑制伴放线放线杆菌(A.a)和牙龈二氧化碳噬纤维菌(C.g)生长效果。方法:应用免疫接种法、水稀释法、盐析法、液体培养抑菌法、以及ELISA法,诱导、提取和纯化IgY抗体,取一定量抗体与细菌共同培养,测定抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长效果。结果:两步硫酸铵盐析沉淀的IgY抗体纯度达85.6%～90.3%;抗原结合效价为1∶32000;抗伴放线放线杆菌IgY抗体与牙龈二氧化碳噬纤维菌交叉免疫反应的抗原结合效价为1∶8000;当抗伴放线放线杆菌IgY抗体浓度在5.0、1.0、0.1g/L时,细菌浓度在5×108CFU/L培养24h其抑菌率分别为31.60%(P=0.004)、10.24%(P=0.024)、-3.30%,培养72h其抑菌率分别为64.20%(P=0.004)、53.21%(P=0.002)、11.20%。细菌浓度在1×108CFU/L培养24h其抑菌率分别为35.71%(P=0.004)、30.95%(P=0.012)、11.11%,培养72h其抑菌率分别为65.11%(P=0.005)、54.04%(P=0.002)、16.17%;5.0g/L的抗伴放线放线杆菌IgY与1×108CFU/L牙龈二氧化碳噬纤维菌培养24h其抑菌率为41.61%(P=0.005),培养72h抑菌率为86.99%(P=0.014)。结论:伴放线放线杆菌能够诱导母鸡产生高效价的特异性IgY抗体,该抗体在一定的浓度内有抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长的作用;伴放线放线杆菌与牙龈二氧化碳噬纤维菌存在着共同抗原。     

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℃…     

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。     

静磁场对口腔变形链球菌生长影响体外实验

目的:研究磁性附着体静磁场对口腔变形链球菌生长的影响.方法:将口腔变形链球菌分为12.5 mT和120 mT强度的静磁场加载组以及未加载磁场对照组,厌氧培养20 h,从培养后第8 h开始,每2 h进行活菌计数.结果:12.5 mT静磁场组变形链球菌活菌数与对照组相比未见显著差异(P>0.05),120 mT静磁场组细菌...     

血链球菌细菌素对伴放线聚集杆菌和白色念珠菌生物膜作用的研究

目的:研究血链球菌细菌素分别对单独培养及混合培养的伴放线聚集杆菌(A.a)和白色念珠菌(C.a)生物膜的最小抑菌浓度(MIC)。利用激光共聚焦显微镜研究血链球菌细菌素对A.a及混合培养的A.a和C.a生物膜活性的影响。方法:通过低温高速离心,细胞破碎,盐析等方法提取血链球细菌素—血链素。采用二倍稀释法,测定血链素对单独和混合培养的A.a及C.a的MIC;血链素作用于A.a及混合培养的A.a和C.a后,利用激光共聚焦显微镜分别在2 h、6 h、12 h、24 h、48 h、72 h观察生物膜活性的变化。结果:血链素对单独培养的A.a的MIC为0.25 g/L;血链素对单独培养的C.a的MIC为0.125 g/L;血链素对混合培养的A.a及C.a的MIC为0.5 g/L。通过激光共聚焦显微镜观察,血链素对A.a生物膜活性有较强的抑制作用,最显著的时间在72 h;血链素对混合培养的A.a及C.a生物膜活性同样具有较强的抑制作用,最显著的时间在48 h。结论:血链素对A.a及混合培养的A.a及C.a生物膜活性均有抑制作用。     

静磁场对口腔变形链球菌生长影响体外实验

目的：研究磁性附着体静磁场对口腔变形链球菌生长的影响。方法：将口腔变形链球菌分为12.5 mT和120 mT强度的静磁场加载组以及未加载磁场对照组,厌氧培养20 h,从培养后第8 h开始,每2 h进行活菌计数。结果：12.5 mT静磁场组变形链球菌活菌数与对照组相比未见显著差异（P〉0.05）,120 mT静磁场组细菌活菌数低于对照组（P〈0.05）。结论：12.5 mT静磁场对口腔变形链球菌生长无影响,120 mT静磁场抑制变形链球菌的生长。     

臭氧水对伴放线放线杆菌的灭活效果观察

目的探讨臭氧水对牙周可疑致病菌伴放线放线杆菌（Aa）的灭活效果。方法采用悬液定量杀菌方法和和化学方法在实验室进行观察。用4、8、15mg／L的臭氧水分别对悬液中A。作用1、2、3min。结果当臭氧水浓度为4mg／L对悬液中An没有杀灭作用。当臭氧水浓度为8mg儿时,对悬液中An作用1min,杀灭率为57％,当浓度上升至15mg／L时,对悬液中Aa作用1min．杀灭率上升至98％．而延长杀菌时间至3min．杀菌率维持在97％～99％。结论在25℃的室温条件下．臭氧水浓度达到15mg／L．臭氧水温控制在15℃～18℃时对悬液中Aa有快速、有效的杀灭作用。     

Aminopeptidase activity in strains of oral streptococci

Ten strains of oral streptococci were analyzed for aminopeptidase activity by isoelectric focusing and crossed immunoelectrophoresis together with enzyme staining procedures. Substrate specificities were determined using 15 aminoacyl-β-naphthylamides. All 10 strains tested showed enzyme activity with the arginine and leucine substrates. None of the strains exhibited activity with the glycine, cystine, or proline substrates. Some strains had multiple enzyme bands with some of the substrates. All strains had aminopeptidase bands located in the pi range 4.1–4.3, except Streptococcus mutans IB and Streptococcus milleri NCTC 10708. The isoelectric profiles of these strains, having enzyme bands in the range 4.45–4.9, differed markedly from the others. These were also the only strains where no arginine aminopeptidase cross-reactivity occurred with the Streptococcus mitis ATCC 903 antiserum.     

Penicillin-induced lysis in related species of oral streptococci

Treatment of 10 strains representing 5 species of related oral streptococci with penicillin G resulted in measurable levels of cellular lysis. This lysis was dependent on penicillin concentration and cell density. At similar penicillin concentrations and cell densities, the 10 strains demonstrated appreciable differences in their lytic responses. Lysis values for Streptococcus mutans (GS-5 and Ingbritt) and Streptococcus rattus (BHT and FA-1) were greater than those for Streptococcus sobrinus (01 and B13), Streptococcus cricetus (HS6 and AHT), and Streptococcus ferus (8S1 and HD3).     

Membrane locus and pH sensitivity of paraben inhibition of alkali production by oral streptococci

Parabens were found to be potent inhibitors of alkali production from arginine by oral streptococci such as Streptococcus rattus, Streptococcus sanguis and Streptococcus gordonii. For example, 2 mumol butylparaben per ml completely and irreversibly inhibited arginolysis by intact cells of S. rattus FA-1 and was lethal for the organism. In contrast, butylparaben was not a very effective inhibitor of ureolysis by intact cells of Streptococcus salivarius 57.I, although it did kill the cells. Butylparaben irreversibly inhibited the cytoplasmic enzymes arginine deiminase, carbamate kinase and urease in permeabilized cells or isolated form. However, inhibition of arginolysis by intact cells appeared to be due primarily to irreversible inhibition of transport systems for arginine uptake, because butylparaben added to intact cells did not reduce levels of arginine deiminase when the cells were subsequently permeabilized after washing. The insensitivity of ureolysis by intact cells to butylparaben can be related to the known high permeability of cell membranes to urea and the cytoplasmic location of urease. The potency of butylparaben as an inhibitior of arginolysis or glycolysis and as a lethal agent was found to be greater at acid pH that at neutral or alkaline pH.     

Ecology of viridans streptococci in the oral cavity and pharynx

Recently published taxonomic studies of viridans streptococci have resulted in several changes in the nomenclature and definition of oral streptococcal species. With this background, the ecology of streptococci in the oropharyngeal cavities was reinvestigated. The results based on the examination of 1426 streptococcal isolates confirmed and extended earlier findings. Apart from mature supragingival plaque, which contained a mixture of all orally encountered streptococci, each site showed a characteristic streptococcal flora. Initial dental plaque formation is primarily associated with Streptococcus sanguis, Streptococcus mitis biovar 1 and Streptococcus oralis. Our investigation showed that S. sanguis and S. mitis biovar 1 were the most prominent streptococci, also on buccal mucosa. In contrast, S. oralis was almost exclusively found in initial dental plaque. Streptococcus gordonii, formerly part of S. sanguis, was found in small numbers on the oropharyngeal mucosa and in mature supragingival plaque. The dorsum of the tongue was dominated by S. mitis biovar 2 and Streptococcus salivarius, the latter of which was predominant also on the pharyngeal mucosa. Streptococcus anginosus was by far the most predominant streptococcus in subgingival plaque. Immunoglobulin A1 (IgA1) protease-producing streptococci were primarily isolated from initial dental plaque and from the buccal mucosa. This lends further support to the concept of IgA1 proteases being important for the ability of streptococci to evade the local immune defence during their initial colonization of certain oral surfaces.     

Bactericidal action of tachyplesin I against oral streptococci

Tachyplesin I, a polycationic antimicrobial peptide isolated from hemocytes of horseshoe crabs, kills bacteria by disrupting the membrane potential of the cytoplasmic membrane. The present study shows that, among 36 oral streptococcal strains, 12 of 21 Streptococcus sanguis , 3 Streptococcus mutans , 9 Streptococcus salivarius and 3 Streptococcus milleri strains were susceptible to tachyplesin I, whereas 9 S. sanguis strains were resistant. Interestingly, these resistant strains include the clinical isolates from both Kawasaki disease and Behget patients. According to the time-kill study, tachyplesin I inhibited irreversibly the growth of S. sanguis. S. tuutans and S. salivarius strains within 20 min and an S. milleri strain within 20 min. Although it has been suggested that Escherichia coli cultured in rich media were more susceptible to tachyplesin I, the present results show thai only 3 S. milleri strains were more sensitized to tachyplesin I in a glucose-supplemented medium, and other tested strains were not. Similarly, only 4 strains were more resistant to tachyplesin I in saline than these were in a rich medium.     

Effect of inorganic ions and surface active organic compounds on the adherence of oral streptococci

abstract — A reduction in the adherence of oral streptococci was observed after topical application of aluminum ions to standardized dentin test pieces whereas pyrophosphate and tripolyphosphate seemed to inhibit the adherence after incorporation in the incubation solution. A pronounced effect was recorded after topical application of surfactants with the positively charged primary amino group as end group. Hydrofluorides of several such aliphatic amines effectively reduced the number of colony-forming units washed off from the test pieces. Also the free base of hexadecylamine showed a similar effect. Inhibition of adherence was also obtained with long chain esters of lysine, with an optimum at 16 carbon atoms in the alcohol group. These compounds have the advantage of being built up from molecules known to the body.     

Effect of low-molecular-weight chitosans on the adhesive properties of oral streptococci

It was previously shown that a low-molecular-weight chitosan and its derivatives N-carboxymethyl chitosan and imidazolyl chitosan inhibit Streptococcus mutans adsorption to hydroxyapatite. The ability of the same molecules to interfere with adhesive properties of other oral streptococci (Streptococcus sanguis, Streptococcus gordonii, Streptococcus constellatus, Streptococcus anginosus, Streptococcus intermedius, Streptococcus oralis, Streptococcus salivarius, Streptococcus vestibularis) was tested. When saliva-coated or -uncoated hydroxyapatite beads were treated with N-carboxymethyl chitosan, a reduction varying from 60% to 98% depending on strains was observed. Low-molecular-weight chitosans and imidazolyl chitosan did not have any effect. Growth in N-carboxymethyl chitosan-supplemented medium (final concentrations ranging from 20 to 500 micrograms.ml-1) caused a dose related reduction in the adsorption of all strains to hydroxyapatite and in their affinity towards xylene. No effect was observed with low-molecular-weight chitosans and imidazolyl chitosan. In contrast to what observed with S. mutans, the three polysaccharides did not affect detachment from hydroxyapatite beads and adherence to cheek epithelial cells of the other streptococci. These results suggest that low-molecular-weight chitosans and/or imidazolyl chitosan, selectively affecting S. mutans adsorption to hydroxyapatite, may be very interesting as potential anti-dental caries agents.     

Antimicrobial activity of garlic against oral streptococci

Abstract:  The antimicrobial activity of two garlic clones' (1: purple and 2: white) crude extracts against oral microbiota was evaluated in vitro (study 1) and in vivo (study 2). Study 1 consisted of the evaluation of minimum inhibitory (MIC) and bactericidal (MBC) concentrations against nine streptococci strains. In study 2, a 2.5% garlic (clone 2) solution was used as a mouthwash in a 5-week study by 30 subjects. Blood agar and Mitis Salivarius Bacitracin agar were inoculated with subjects' saliva to quantify oral microorganisms and mutans streptococci. Study 1 showed MIC ranging from 0.5 to 32.0 mg ml⁻¹ for clone 2 and from 8 to 64.0 mg ml⁻¹ for clone 1. MBC ranged from 1.0 to 128.0 mg ml⁻¹ and from 8.0 to 128.0 mg ml⁻¹ regarding clones 2 and 1 respectively. Study 2 showed that 2.5% garlic mouthwash solution had good antimicrobial activity against mutans streptococci and oral microorganisms. Maintenance of reduced salivary levels of streptococci was observed after 2 weeks at the end of mouthwash use. Unpleasant taste (100%), halitosis (90%) and nausea (30%) were reported by subjects after the end of the study. It was concluded that the garlic clones have antimicrobial properties in vitro against streptococci and anticariogenic properties against oral microorganism in spite of its adverse effects.     

Effects of pulsing with xylitol on mixed continuous cultures of oral streptococci

Continuous culture is a means whereby organisms can be grown at rates approaching those occurring naturally. Moreover, the effect of adding transient excesses of various nutrients to the culture vessel ('pulsing') simulates the effect of dietary challenge on dental plaque organisms. Mixed cultures of Streptococcus mutans T8 and Streptococcus milleri B448 were grown glucose-limited in a chemically defined medium under an atmosphere of 5 per cent carbon dioxide in nitrogen, at a dilution rate of D = 0.1 h-1 and controlled pH of 7.0. The level of arginine in the medium reservoir was adjusted so that Strep. milleri predominated over Strep. mutans in a stable coexistence. After equilibration, the culture vessel was pulsed with various carbohydrates to a final concentration of 5 x 10(-2)mol/L. Samples were then taken at regular intervals and differential viable counts of Strep. mutans and Strep. milleri were done on mitissalivarius agar. Results demonstrated that pulsing with glucose, fructose, 'coupling sugar', lactose, xylose and sorbitol gave Strep. mutans a clear ecological advantage. In direct contrast, pulsing with xylitol resulted in a marked antimicrobial effect on Strep. mutans while Strep. milleri was essentially unaffected. This supports recent findings by other workers that uptake of this pentitol by Strep. mutans in batch culture sets up a 'futile cycle', leading to depressed growth or even cell death.     

Inhibitory effects of cranberry juice on attachment of oral streptococci and biofilm formation

Cranberry juice is known to inhibit bacterial adhesion. We examined the inhibitory effect of cranberry juice on the adhesion of oral streptococci strains labeled with [3H]-thymidine to saliva-coated hydroxyapatite beads (s-HA). When the bacterial cells were momentarily exposed to cranberry juice, their adherence to s-HA decreased significantly compared with the control (P < 0.01). Their hydrophobicity also decreased dependently with the concentration of cranberry juice. We also evaluated the inhibitory effect of cranberry juice on biofilm formation. By using a microplate system, we found that the high molecular mass constituents of cranberry juice inhibited the biofilm formation of the tested streptococci. The inhibitory activity was related to the reduction of the hydrophobicity. The present findings suggest that cranberry juice component(s) can inhibit colonization by oral streptococci to the tooth surface and can thus slow development of dental plaque.