葡萄糖浓度影响寡发酵链球菌抑制变形链球菌作用的研究

目的 研究不同葡萄糖浓度对寡发酵链球菌(Streptococcus oligofermentans,So)与变形链球菌(Streptococcus mutans,Sm)之间相互作用的影响,及对So产过氧化氢能力的影响.方法通过平板培养法观察在不同葡萄糖浓度下So与Sm之间的相互作用;运用4-氨基安替吡啉-辣根过氧化物酶法测定不同葡萄糖浓度下So过氧化氢的初始产生速率和产量.结果环境葡萄糖浓度为0、10、50 mmol/L时均可见So对Sm有抑制作用;Sm受抑制区面积占菌膜面积比值:同时接种So和Sm时,无糖环境比值为0.202±0.005,10、50 mmol/L葡萄糖环境分别为0.467±0.025和0.468±0.028;先接种So再接种Sm时,无糖环境比值为0.394 ±0.004,10 mmol/L葡萄糖环境为0.811 ±0.075,50 mmol/L葡萄糖环境为0.816 ±0.007.葡萄糖浓度为10、50 mmol/L时So对Sm的抑制作用均较无糖环境下显著(P＜0.05),但两种浓度抑制作用差异无统计学意义(P＞0.05).葡萄糖浓度为10、50 mmol/L时So的过氧化氢初始产生速率[(23.573±0.263)、(23.337±0.473) μmol·L-1·min -1]均显著高于无糖环境[(10.513 ±0.516) μmol·L-1·min-1],P ＜0.05.无糖环境下So对数生长期各时段过氧化氢产量高于有糖环境(P＜0.05).1000 mmol/L葡萄糖环境下未见So抑制Sm,亦未能检测到So产生过氧化氢.结论So抑制Sm的能力受糖环境的影响,在10、50 mmol/L葡萄糖环境下,So具有更强的抑制Sm能力.     

环境氧条件影响寡发酵链球菌抑制变形链球菌作用的研究

目的 探讨外界环境氧气条件的改变对寡发酵链球菌(Streptococcus oligofermentans,So)与变形链球菌(Streptococcus mutans,Sm)之间抑制作用的影响,以及对So产过氧化氢能力的影响,以期探讨这两种细菌在不同口腔环境条件下的共存模式.方法 通过二氧化碳培养法建立细菌培养的有氧和厌氧环境;运用平板培养法观察So与Sm之间的抑制作用;运用4-氨基安替吡啉-辣根过氧化物酶法测定有氧和厌氧环境下So对数生长期各时间段过氧化氢的产量和So生长周期过氧化氢初始产生速率.结果 同时接种So和Sm,厌氧环境下未见So抑制Sm,有氧环境下So轻度抑制Sm,受抑制区面积约为菌膜面积的1/5;先接种So再接种Sm,两种环境下均可见So抑制Sm,厌氧环境下受抑制区面积约为菌膜面积的1/5,有氧环境下约为4/5;先接种Sm再接种So,两种环境下均未见So生长;有氧环境下So对数生长期各时间段过氧化氢产量明显高于厌氧环境(P<0.05);厌氧环境下So生长周期过氧化氢初始产生速率为每分钟(11.84±3.97) μmol/L,仅为有氧环境时[(24.13±4.46) μmol/L]的49%(P<0.05).结论 环境氧条件影响So对Sm的抑制作用,有氧环境下抑制作用更强;有氧环境下So产过氧化氢的能力强于厌氧环境.

Abstract：

Objective To investigate the effect of environmental oxygen on the inhibition between Streptococcus oligofermentans(So) and Streptococcus mutans(Sm) and the producibilities of hydrogen peroxide by So. Methods The aerobic and anaerobic environment was established by the carbon dioxide cultivation. The inhibition between So and Sm was observed by plating method. The production and synthesis rates of hydrogen peroxide by So were determined in both aerobic and anaerobic environment by 4-ATTP-horseradish peroxidase method at A510. Results When both Sm and So were inoculated at the same time, Sm was not inhibited under the anaerobic environment, vice versa. Sm was slightly inhibited by So under the aerobic environment, the inhibition area was 1/5 of all bacterial membrane. When So was cultivated first and then Sm applied, So could inhibite Sm growth under both anaerobic and aerobic conditions. The inhibition area was 1/5 of bacterial membrane under the anaerobic environment, and 4/5 under the aerobic environment. When Sm was cultivated first and then So applied, So was unable to proliferate under both conditions. During the logarithmic phase, the production of H2O2 by So under the aerobic environment was higher than under the anaerobic environment (P<0.05).The initial synthesis rate of H2O2 by So during growth cycle under the anaerobic condition was (11.84±3.97) μmol/L per minute, which was only 49% of that under the aerobic environment[(24.13±4.46) μmol/L per minute].Conclusions The oxygen has the effect on the inhibition between So and Sm, and the inhibition in the aerobic environment is much stronger than in the anaerobic environment. The synthesis ability of hydrogen peroxide by So under the aerobic environment is higher than under the anaerobic environment.     

寡发酵链球菌乳酸脱氢酶蛋白质结构的预测

目的 探讨寡发酵链球菌和口腔中不同产酸能力的细菌乳酸脱氢酶(lactatedehydrogenase,LDH)基因序列和蛋白质各级结构的差异,为进一步研究寡发酵链球菌糖代谢的生化机制提供依据.方法 寡发酵链球菌LDH的基因序列由中科院微生物所测出.通过美国国家生物技术信息中心的Genbank数据库查找变形链球菌、血链球菌和干酪乳杆菌LDH的基因序列,用BLAST软件对各细菌LDH的基因序列和氨基酸序列进行比对.采用蛋白分析专家系统(Expert Protein Analysis System,ExPASy)对寡发酵链球菌、变形链球菌、血链球菌和干酪乳杆菌LDH蛋白质的理化性质、二级结构、结构域和空间结构进行预测并比较.结果 寡发酵链球菌LDH的基因序列共987个碱基对,与血链球菌相似性最高(89％),与变形链球菌的相似性为81％,与干酪乳杆菌相似性最低(70％);寡发酵链球菌LDH氨基酸序列与血链球菌的相似性高达96％,与变形链球菌相似性较低(86％),与干酪乳杆菌的相似性仅为66％;寡发酵链球菌LDH蛋白质的理化性质、跨膜区数目、二级结构的比例、结构域位置与其他3种细菌均相近,LDH的空间结构与变形链球菌、干酪乳杆菌明显不同,与血链球菌也有差异.结论 寡发酵链球菌LDH在基因序列、氨基酸序列及空间结构上与变形链球菌和干酪乳杆菌的差异,可能是导致其LDH生物学功能改变,不能产生乳酸的一个内在原因.     

蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响

目的探讨蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响,并与血链球菌和变异链球菌的双菌种生物膜形成进行比较。方法运用菌落计数法观察蔗糖环境下唾液包被的玻璃生物模型中单/双菌株寡发酵链球菌,变异链球菌和血链球菌24 h生物膜形成情况;运用激光共聚焦显微镜观察蔗糖环境下寡发酵链球菌,变异链球菌和血链球菌单/双菌株生物模型中24h生物膜形成厚度。结果无糖环境下,单菌株模型中,菌落数:血链球菌(55.67±5.36)>变异链球菌(53.48±2.63)(P>0.05)>寡发酵链球菌(46.24±2.34)(P<0.05);生物膜厚度:血链球菌(17.23±3.82)>变异链球菌(15.16±4.21)(P>0.05)>寡发酵链球菌(10.54±4.37)(P<0.05)。双菌株模型中,变异链球菌菌落数降低幅度:血链球菌组>寡发酵链球菌组(P<0.05)。生物膜厚度:血链球菌组(8.12±2.82)<寡发酵链球菌组(11.27±3.55)(P<0.05)。蔗糖环境下,单菌株模型中,菌落数:变异链球菌(58.54±2.74)>血链球菌(51.87±5.35)>寡发酵链球菌(48.57±3.05)(P<0.05)生物膜厚度:变异链球菌(20.63±5.71)>血链球菌(13.37±4.93)>寡发酵链球菌(12.45±4.62)(P<0.05)双菌株模型中;变异链球菌菌落数降低幅度,寡发酵链球菌组>血链球菌组(P<0.05),生物膜厚度:寡发酵链球菌组(6.67±2.19)<血链球菌组(10.45±2.72)(P<0.05)。结论外界糖环境影响寡发酵链球菌和血链球菌对变异链球菌的抑制作用,蔗糖环境下,寡发酵链球菌的抑制作用强于血链球菌。     

壳寡聚糖对变形链球菌黏附的影响

目的:研究壳寡聚糖对变形链球菌蔗糖依赖性黏附的影响。方法:使用不同浓度的壳寡聚糖与变形链球菌S.mutansATCC25175共同培养,计算壳寡聚糖对变形链球菌的黏附抑制率的影响,同时扫描电镜下观察变形链球菌在玻片的形态学特征。结果:变形链球菌对玻片的黏附率随培养基中壳寡聚糖浓度的升高而下降。扫描电镜图片显示壳寡聚糖组细菌间基质少,菌细胞清晰。而空白组细菌则包裹有较多的胶冻状基质。结论:壳寡聚糖可以有效抑制变形链球菌的蔗糖依赖性黏附。     

壳寡聚糖对变形链球菌表面疏水性的影响

目的:研究壳寡聚糖对变形链球菌细胞表面疏水性的影响,从而进一步了解壳寡聚糖影响细菌黏附的机制。方法:使用不同浓度(10-2、10-1、1g/L)的壳寡聚糖溶液处理变形链球菌,并采用微生物黏着碳氢化合物法测试细胞表面疏水性。结果:随壳寡聚糖浓度增加,变形链球菌ATCC25175细胞表面疏水性有显著性降低。结论:壳寡聚糖可能通过降低细菌表面疏水作用而抑制变形链球菌的黏附。     

壳寡聚糖抑制变形链球菌的致龋能力

目的研究壳寡聚糖(chitooligosaccharide,COS)对变形链球菌生长、产酸及粘附能力的影响。方法选用变形链球菌标准株ATCC 25175,采用对倍稀释法测定COS对变形链球菌的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC);采用菌落计数法,通过时间—杀菌曲线的变化,分析COS对细菌生长速度的影响;将COS及变形链球菌菌悬液各1 m L接种于无菌试管内厌氧培养,使COS的终浓度分别达到1/2 MIC、1/4 MIC、1/8MIC,测定上清液p H变化并绘制p H曲线;采用液体闪烁计数法测定壳寡聚糖对变形链球菌粘附羟磷灰石能力的影响。结果 COS对变形链球菌的MIC和MBC分别为2.00 g/L和4.00 g/L;COS能有效抑制变形链球菌的生长,较高浓度时对细菌还具有杀菌作用。亚抑菌浓度的COS可显著影响变形链球菌的产酸性和粘附能力,并且这种抑制作用呈浓度依赖性。结论 COS对变形链球菌的生长、产酸和粘附能力均有抑制作用。     

经放射线照射后口腔链球菌的分子生物学鉴定

通过生理、生化试验可对口腔及咽喉部的97．9％的口腔链球菌分离株做出鉴定。但是对一些生长缓慢的细菌，或表型特征介于两个种之间的菌株，只靠生化试验不能明确鉴定到种。而分子生物学鉴定方法如16S rDNA序列同源性分析可将细菌准确、快速地鉴定到种。我们采用16S rDNA序列同源性分析的方法对放疗后生化试验表型发生变化的菌     

颗粒链球菌属的研究进展

颗粒链球菌属是兼性厌氧、触酶阴性的革兰阳性球菌,口腔微生物组研究发现颗粒链球菌属是人口腔的优势菌,可引起牙周病、牙髓炎等机会感染.本文重点介绍颗粒链球菌属的最新研究进展.     

颗粒链球菌属的研究进展

颗粒链球菌属是兼性厌氧、触酶阴性的革兰阳性球菌,口腔微生物组研究发现颗粒链球菌属是人口腔的优势菌,可引起牙周病、牙髓炎等机会感染.本文重点介绍颗粒链球菌属的最新研究进展.     

Comparison between a micromethod and a conventional method for estimation of salivary Streptococcus mutans

Abstract – Two methods for estimating the number of S. mutans in 501 paraffin-wax stimulated whole saliva samples were compared. A highly significant correlation was found between the results of a micropipette method and conventional agar plating. The micropipette method is simple, reliable and economical and is a suitable alternative to conventional plating for determination of the S. mutans infection level.     

A method for studying adherence of oral streptococci to solid surfaces

abstract — The adherence of different streptococci to test pieces of glass, human enamel and whale dentin carried in the mouth or immersed in saliva or bacterial suspensions was studied by examining the number and frequency of microorganisms which were selectively desorbed by a standardized washing technique. The results obtained from in vivo and in vitro experiments were similar in principle. The proportion of streptococci obtained in the first washing resembled that found in the saliva, but with more vigorous washing the proportion of S. sanguis increased while that of S. salivarius decreased. This illustrates that different microorganisms can attach to solid surfaces with different strengths. The applicability of the method was tested by treating dentin surfaces with fluoride solutions and by incorporating sucrose in the test solution. The fluoride treatment reduced while the sucrose addition increased the number of streptococci which could be removed from the surfaces.     

Association of a novel high molecular weight, serine-rich protein (SrpA) with fibril-mediated adhesion of the oral biofilm bacterium Streptococcus cristatus

The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 +/- 19 nm long with longer, less abundant fibrils 403 +/- 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 +/- 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 +/- 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.     

Association of a novel high molecular weight,serine‐rich protein (SrpA) with fibril‐mediated adhesion of the oral biofilm bacterium Streptococcus cristatus

The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 ± 19 nm long with longer, less abundant fibrils 403 ± 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 ± 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 ± 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild‐type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine‐rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram‐positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial‐associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.     

Development of an animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study

Background: Biofilm‐induced inflammatory osteolytic oral infections, such as periodontitis and peri‐implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants. Methods: Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm‐inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri‐implant bone volume. Results: Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm‐inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100% of biofilm‐inoculated implants for up to 3 weeks and 25% for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm‐inoculated implants (29.6% ± 7.6%) compared to non‐inoculated implants (50.5% ± 9.6%) after 6 weeks. Conclusions: These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.     

Detection and quantification of Aggregatibacter actinomycetemcomitans,Porphyromonas gingivalis and Streptococcus oralis in blood samples with different microbiological identification methods: An in vitro study

BackgroundCulture-based methods (culture broth bottles or lysis methods) have been the standard for detecting bacteremia. More recently, quantitative polymerase chain reaction (qPCR) was proposed as a more sensitive and specific test although none of them has been validated for the identification of periodontal pathogens (fastidious growing bacteria) in blood samples.ObjectiveTo compare the ability to detect and quantify Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus oralis (alone or in combination) in blood samples with three culture techniques [direct anaerobic culturing (DAC), haemo-culture (BACTEC), and lysis-centrifugation (LC)] and a non-culture dependent approach (qPCR) in an in vitro study.Material and methodsBlood samples from 12 periodontally healthy volunteers were contaminated with three concentrations [10⁴,10² and 10¹ colony forming units (CFU)/mL] of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination. Samples were analysed by DAC, BACTEC, LC and qPCR. Sensitivity, specificity, predictive values, kappa index and Lińs correlation coefficients were calculated.ResultsDAC, LC and qPCR were able to detect the three target species at all concentrations. An excellent concordance (correlation coefficient r: 0.92–1) was observed between DAC and the reference standard (sensitivity raging 93.33–100% and specificity 88.89–100%) values. BACTEC was not able to identify P. gingivalis in any of the performed experiments. qPCR provided false negative results for S.oralis.ConclusionsDAC showed the best results for the proper identification and quantification of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination, in blood samples.     

Transient acid‐impairment of growth ability of oral Streptococcus,Actinomyces, and Lactobacillus: a possible ecological determinant in dental plaque

Introduction: Dental plaque pH decreases to about 4 through bacterial fermentation of carbohydrates and this low pH is maintained for from several minutes to about an hour. Repeated acidification causes demineralization of the tooth surface, resulting in caries formation. The acidification also influences plaque bacteria. Severe acidification kills bacteria efficiently, while physiological acidification, the condition occurring in plaque, kills bacteria partially and may impair growth ability. We, therefore, investigated the effects of physiological acidification on representative caries‐related bacteria. Methods: Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, Streptococcus oralis, Lactobacillus paracasei, and Actinomyces naeslundii were used. Effects of physiological acidification at pH 4.0 on cell viability and growth ability, as well as the growth rate of these bacteria at pH 4.0–7.0, were investigated. Results: Mutans streptococci and Lactobacillus grew at pH 4.0 but the growth of S. sanguinis and S. oralis ceased below pH 4.2 and pH 4.2–4.4, respectively. Acidification at pH 4.0 for 1 h killed 43–89%, 45% and 35–76% of S. sanguinis, S. oralis, and Actinomyces, respectively. Furthermore, assessment of bacterial growth curves revealed that the growth ability of the surviving cells of S. sanguinis, S. oralis and Actinomyces was impaired, but it was recovered within 2–5 h after the environmental pH had returned to 7.0. The acidification neither killed nor impaired the growth of mutans streptococci and Lactobacillus. Conclusions: These results indicate that physiological and transient acidification is not sufficient to kill bacteria, but it causes a temporary acid‐impairment of their growth ability, which may function as an ecological determinant for microbial composition in dental plaque.     

T‐RFLP‐based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype

Introduction: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. Methods: Forty‐four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real‐time quantitative polymerase chain reactions. Results: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non‐synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl‐coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. Conclusion: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.     

Satisfaction of patients with amyotrophic lateral sclerosis with an oral appliance for managing oral self-biting injuries and alterations in their masticatory system: A case-series study

Statement of problem

About 10% of patients with amyotrophic lateral sclerosis (ALS) are candidates for oral treatment specifically because of traumatic injuries in the lips, cheeks, or tongue due to self-biting. However, patients with ALS have a prevalence of temporomandibular disorder (TMD) similar to that in the general population.