Synergistic degradation of bovine serum albumin by mutans streptococci and other dental plaque bacteria

Mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) exhibited low levels of proteolytic activity against the model protein substrate, FITC-labelled bovine serum albumin, when incubated alone. Inclusion of other members of the dental plaque microflora in the assay usually resulted in marked increases in the degree of proteolysis and a high level of synergy. Interactions between mutans streptococci and either Streptococcus oralis or Fusobacterium nucleatum gave rise to the greatest degree of synergistic proteolytic degradation.     

Biogenesis of methane in primate dental plaque

Dental plaque samples collected from monkeys (Macaca mulatta) were found to contain a large amount of dissolved methane gas (0.6 nmol CH4/mg wet wt plaque). Enrichment cultures inoculated with dental plaque obtained from Macaca fascicularis produced methane when the medium contained ethanol, methanol, lactate, acetate or a hydrogen + CO2 atmosphere. Methane formation in the enrichments was inhibited by oxidation of the culture medium, autoclaving or the addition of 2-bromoethane sulfonic acid (BES). The methane producing enrichments were observed to contain fluorescent cocci occurring singly and in short chains. It was concluded that methane formation in the monkey dental plaque was the result of the presence of methanogenic bacteria.     

Competition for dietary carbohydrates between streptococci in dental plaque

Abstract The predominant microorganisms in dental plaque, i.e., streptococci and actinomycetes, are carbohydrate fermenters. In the natural environment these organisms experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. Recently, we have studied the competition between pairs of oral Streptococcus species in the chemostat under continuous glucose or sucrose limitation and under glucose pulsed conditions.
In the present study we have investigated the competition for dietary carbohydrates in dental plaque of gnotobiotic rats infected with the same combinations of streptococci. The rats were exposed to similar regimes of carbohydrate administration as in the chemostat by incorporation of low molecular carbohydrates in the diet and drinking water. Addition of glucose to the diet favoured the organism with the highest q _max for glucose. This result parallels the outcome of earlier competition experiments in glucose-pulsed chemostats, which showed that the organism with the highest q _max glucose became dominant in the cultures. Previous observations that bacteriocin production and extracellular glucan synthesis are major ecological factors for oral streptococci were also confirmed in this experiment.     

牙斑幽门螺杆菌与慢性胃炎之间的关系

目的研究牙斑幽门螺杆菌与慢性胃炎之间的关系。方法对胃炎组、胃炎治疗组分别进行牙斑和胃黏膜幽门螺杆菌培养和比较。结果牙斑细菌培养：胃炎组阳性14例,阳性率为12．8％;治疗组阳性11例,阳性率为10．1％。胃黏膜细菌培养：胃炎组阳性47例,阳性率为43．1％;治疗组阳性19例,阳性率为17．4％。治疗前后比较牙斑标本差异无显著性（P〉0．05）,胃黏膜标本差异有非常显著性（P〈0．001）。结论牙斑中确实存在着幽门螺杆菌,而且是胃内反复感染的源泉,以致慢性胃病反复发作,难以治愈。     

群体感应(QS)在牙菌斑形成中的研究进展

形成牙菌斑的生物膜中存在大量微生物,各种微生物通过高丝氨酸内酯(AHL)或寡肽等不同的信号分子产生群体感应(QS),形成的群体感应使各种微生物建立了区系平衡,对龋齿及牙周炎等口腔疾病的治疗产生严重影响。因组成生物膜的各种微生物对抗生素的敏感性和耐受性有显著差异,因此在治疗中增加了对宿主免疫的相应要求。为进一步探讨QS系统在牙菌斑形成中的作用特点,本文就牙菌斑的形成与抗药机制、群体感应系统及其信号分子、群体感应系统的抑制因子和研究展望进行综述。     

Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis

A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease. One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii. These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced. M. oralis was found to be the predominant archaeon in the subgingival dental plaque. Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp. within the Methanobacteriaceae family.     

牙菌斑形成早期微生物定植规律的定量PCR研究

目的观察牙面彻底清洁后24 h内牙面上定植的变异链球菌、伴放线放线杆菌和总微生物的数量变化。方法 8名健康成人接受全口洁治后,分别于6、12和24 h收集龈上菌斑,提取菌斑内细菌的基因组DNA。设计变异链球菌、伴放线放线杆菌和总菌特异性引物,获得目的基因,克隆于大肠埃希菌DH5α感受态细胞,测序后获得质粒标准品。将样本和梯度稀释的质粒标准品进行SYBR Green I实时荧光定量PCR检测,绘制标准曲线,确定样本中变异链球菌、伴放线放线杆菌和总菌DNA拷贝数。结果牙面彻底清洁6 h后即有大量变异链球菌定植,变异链球菌拷贝数占总菌的0.32%,24 h后增加到0.67%。12 h时定植的变异链球菌拷贝数高于6 h,差异有统计学意义（P=0.031）,24 h后继续增加（P=0.024）。12 h时定植的总菌拷贝数高于6 h,差异有统计学意义（P=0.004）,24 h后继续增加（P=0.042）。牙菌斑中伴放线放线杆菌的拷贝数低于103。结论早期牙菌斑中12 h定植的变异链球菌和总菌数量高于6 h,且24 h内不断增加,仅有少量伴放线放线杆菌定植。     

早晚牙菌斑变异链球菌定植差异的定量PCR检测

目的 采用实时荧光定量PCR检测白天和晚上所形成牙菌斑生物膜中变异链球菌的数量,比较早晚牙菌斑中变异链球菌定植的差异.方法 收集30名健康成人全口口腔洁治后白天和晚上形成的龈上菌斑,提取细菌基因组DNA.合成变异链球菌特异性引物,纯化PCR产物获得目的基因连接于pTA-TA载体,克隆于大肠埃希菌DH5 α感受态细胞.选取阳性克隆测序后纯化质粒DNA,获得质粒标准品.将样本和梯度稀释的质粒标准品进行SYBR Green Ⅰ实时荧光定量PCR检测,确定标准曲线,定量样本中变异链球菌DNA的拷贝数.结果 早晚牙菌斑细菌基因组DNA样本的扩增曲线均在标准品的扩增曲线范围内.晚上所形成牙菌斑中定植的变异链球菌数量(对数值7.67 ±0.77)高于白天定植的变异链球菌数量(对数值7.25±0.62)(P =0.007).结论 牙菌斑的微生物定植存在日夜节奏变化,晚上所形成牙菌斑中变异链球菌数量多于白天.     

Xive种植体周龈下可疑致病菌变化的1年观察

目的研究一年内Xive种植体周常见致病菌的变化情况,为种植体的定期维护提供理论基础。方法临床选取32名种植患者的44枚种植体,记录了修复后1个月,修复后3个月,修复后6个月,修复后12个月四个时段,入选种植牙的菌斑指数（PLI）,牙龈指数（G1）,牙龈出血指数（GBI）,探诊深度（PD）;采用产黑菌、放线菌、具核梭杆菌及厌氧菌的选择性培养基对龈下菌斑标本进行了分离培养。结果随时间延长,种植体周几种龈下菌的检出量除牯放菌外不断增加,1个月到3个月时增加的趋势最为明显,到6个月左右趋于稳定。厌氧菌总数、产黑菌、核梭菌、粘放菌的统计值在1个月和3个月、3个月和6个月比较差异有显著性,但在6个月和12个月比较差异无显著性。各临床指标和X-线指标随时问的变化差异无显著性。结论在临床工作中,应注意加强修复后3个月时种植患者的口腔维护,防止种植体周围炎的发生,提高种植成功率。     

牙菌斑生物膜的形成与控制

牙菌斑生物膜是附着于牙釉质表面,由复杂的微生物群落构成的一种聚集体。牙菌斑生物膜的形成与生长对口腔健康有着直接或间接的影响,许多研究证实口腔疾病如龋齿和牙周病都与细菌的积累及牙菌斑的形成有关。在牙菌斑生物膜形态建成过程中,牙齿表面最初的定殖菌对生物膜的微生物组成和结构至关重要,这些初级定殖菌决定了后续与之结合形成共生体的微生物种类和数量。不同的微生物组成可能在与生物膜形成相关的口腔病理状况中发挥不同的作用。因此,本文就牙菌斑生物膜的生长及控制进行综述,介绍其微生物的早期定殖和成熟过程、以及通过物理和化学方法对牙菌斑生物膜的控制,以期为了解牙菌斑生物膜的形成机制及相关口腔疾病的预防和治疗提供有价值的参考。     

Experimental abscess formation caused by human dental plaque

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess-forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti-phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 x 10(6) colony-forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat-treated supragingival dental plaque influenced the abscess-forming ability of S. anginosus isolate. These isolates possessed a high antiphagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.     

Growth and molecular characterization of dental plaque microcosms

AIMS: (i) To compare the effects of feeding protocols upon the composition and stability of dental plaque microcosms formed in constant-depth film fermenters (CDFF). (ii) To evaluate the utility of denaturing gradient gel electrophoresis (DGGE) and culture methodologies for the investigation of such models. METHODS AND RESULTS: Microcosms were established anaerobically in the CDFFs from freshly collected saliva. These were fed either with artificial saliva alone (famine) or combined with discontinuous feeding (feast-famine). Culture and 16s rDNA sequencing indicated that supplemental feeding gave ca. 2 log increases in Lactobacillus rhamnosus and Prevotella buccae. Feast-famine microcosms were then further characterized by DGGE using primers specific for the V2-V3 region of eubacterial rDNA. These gave single major bands with pure cultures (eight species) and resolved all strains apart from Lact. rhamnosus and Actinomyces naeslundii. Whilst culture with selective media indicated a degree of stability and reproducibility between replicate microcosms, DGGE showed a considerable degree of variability that related to several putatively uncultured bacteria. CONCLUSIONS: Feast-famine regimes altered community composition. DGGE analyses identified putatively unculturable species and demonstrated variability between replicate fermenters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria. Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media. Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics.     

Buffering effect of a prophylactic gel on dental plaque in institutionalised elderly

Objectives: The effect of multiple daily applications of a prophylactic gel, with buffering substances, on plaque acidogenicity in elderly institutionalised individuals was evaluated. Background: Many elderly suffer from reduced salivary flow, poor oral hygiene and increased levels of cariogenic bacteria and are considered to be at an increased risk for coronal and root caries. Reinforcing the buffering capacity of dental plaque by the addition of substances such as bicarbonate and phosphates may decrease their caries activity. Materials and methods: Fourteen elderly, with subjective dry mouth, were treated for 16‐day‐periods at random with: (i) Profylin fluoride gel with buffering components; (ii) Profylin fluoride gel without buffering components and (iii) rinsing with water. Applications were made four times a day and each period was followed by a 2‐week wash‐out period. The plaque pH was registered after a carbohydrate challenge and the following were recorded before and after each test period: stimulated salivary secretion rate, buffer capacity, number Colony Farming Units (CFU) mutans streptococci, lactobacilli and a sample of Candida albicans on oral mucosa. Results: Eleven participants (mean age 76.6 years) fulfilled the study. Changes in plaque pH measurements, when calculated as area under the curve (AUC_6.2 and AUC_5.7) values (pH × min), before and after each of the three treatments, showed no significant differences. A tendency to a higher plaque acidogenicity and amount of cariogenic microorganisms was found after the gel treatments. C. albicans was found in low levels. Conclusion: Frequent applications of the gel did not result in an improved neutralising effect in the elderly. This may be caused by a combination of several factors, such as the level of oral dryness of the individuals and low solubility, release and retention of the gel substances in plaque. Instead, an increased plaque acidogenicity was noted.     

Examiner agreement on caries detection and plaque accumulation during dental surveys of elders

Indices used to evaluate plaque accumulation and coronal caries have been widely accepted in epidemiological studies, yet their reliability cannot be guaranteed. The aim of this study was to evaluate the reliability of clinical criteria used in coronal and root caries diagnosis and oral hygiene evaluation as applied in elders. Nineteen elderly subjects, 73 years old on average, were examined at a first appointment by two independent examiners. They were re-examined two weeks later. Plaque accumulation was evaluated using the Plaque Index (PI) and coronal and root caries were detected according to the WHO criteria and Fejerskov et al (1991), respectively. Recurrent caries was recorded as recommended by WHO and by probing at the interface tooth-restoration. Inter- and intra-examiner agreement was evaluated using kappa statistics. The PI score showed good reliability except for examiner b, for whom a simplification of the 4-point scale in 3-point scale improved significantly the reliability. The prevalence of coronal caries was very low and intra- and inter-examiner agreement was poor. Most of the root caries lesions were covered by plaque and the kappa values indicated only poor agreement. Recurrent caries were found with good agreement using WHO criteria but the detection with the probe was not reliable. In conclusion, it seems that examiners should be trained carefully to maximise their reliability and that plaque should be removed to obtain reliable diagnoses of caries. Retraining and calibration may be necessary for surveys continuing over a long period.     

Extracellular DNA in oral microbial biofilms

The extracellular matrix of microbial biofilms is critical for surface adhesion and nutrient homeostasis. Evidence is accumulating that extracellular DNA plays a number of important roles in biofilm integrity and formation on hard and soft tissues in the oral cavity. Here, we summarise recent developments in the field and consider the potential of targeting DNA for oral biofilm control.     

根表铁氧化物胶膜对水稻吸收Zn的影响

采用营养液培养方法研究了水稻根表形成的铁氧化物胶膜对水稻吸收Ｚｎ的影响．结果表明,在有Ｆｅ２＋的嫌气环境中,由于根际氧化作用水稻根表会形成红色的铁氧化物胶膜,根表的铁氧化物胶膜影响水稻对Ｚｎ的吸收．铁膜数量较少时,由于对Ｚｎ的富集作用有限,其对水稻Ｚｎ的吸收虽有促进作用,但不明显．随着根表铁膜数量的增加,这种促进作用也相应增加,并且在铁膜数量增加到一定值时,对水稻吸收Ｚｎ的促进作用达到最大．而后,随着铁膜数量的进一步增加,铁膜反而阻碍水稻对Ｚｎ的吸收,成为水稻吸收Ｚｎ的障碍层．在此过程中,水稻的根分泌物,特别是其中的植物铁载体对覆有铁膜水稻根系吸收Ｚｎ有促进作用．这种促进作用随铁膜数量的增加而逐渐减弱．因此,根表铁氧化物胶膜对水稻吸收Ｚｎ并不总是起促进作用,其作用的方向和程度取决于铁膜的数量．     

应用PCR与温度梯度凝胶电泳分析龈上菌斑微生物群落

目的应用PCR与温度梯度凝胶电泳(PCR—TGGE)分子技术对成年健康口腔的龈上菌斑中微生物群落组成进行分析。方法8例成年个体包括4男4女,年龄19～29岁,分别采取每例个体上下颌牙周龈上菌斑样品,共18份(个体Subl间隔10天采集2次样品)。提取菌斑DNA,PCR扩增16SrDNAV3可变区,产物经TGGE后进行相似系数分析。结果同一个体的上下颌微生物群落组成相似性系数为81％～95％,而不同个体的龈上菌斑微生物群落组成相似性系数,均在60％以下。结论不同个体具有其独特的牙周微生物群落,而且在一定时期内组成稳定。     

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity

Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface. Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation. Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F. In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis. However, their effect on the growth of S. sobrinus in the solid and liquid phase was notable. 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S. sobrinus in agar plates. Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.     

PCR detection of Capnocytophaga species in dental plaque samples from children aged 2 to 12 years

The purpose of this study was to detect the presence of Capnocytophaga sputigena, C. ochracea, and C. gingivalis in plaque samples from the toothbrushes of 122 children, using a polymerase chain reaction (PCR) method. The subjects were 25, 85, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted tooth sites using a sterile toothbrush. The mean amount of DNA recovered from the samples was approximately 19.3 microg, which was deemed sufficient for performing a PCR-based survey. C. sputigena prevalence in healthy, gingivitis, and periodontitis subjects was 48.0%, 36.5% and 25.0%, respectively, that for C. ochracea was 100%, 89.4%, and 50.0%, respectively, and that for C. gingivalis was 96.0%, 84.7%, and 75.0%, respectively. The lowest age of positive subjects was approximately 2 years. Our results showed that C. sputigena was moderately prevalent, whereas C. ochracea and C. gingivalis were commonly detected in the oral cavities of the tested children, suggesting that all of these species become established in the early years.     

低龄儿童龋病牙菌斑的菌群结构分析

目的通过低龄龋齿儿童与口腔健康儿童的微生物组检测,探讨低龄儿童龋病牙菌斑的菌群组成特征。方法在深圳地区招募轻度龋齿、重度龋齿及口腔健康儿童共100名,采集其牙菌斑或健康牙齿表面微生物样本,采用MiSeq测序平台进行16S V4-V5区域的高通量测序。同时,通过生物信息分析方法,对低龄龋齿儿童的牙菌斑的微生物组成的特征进行挖掘分析。结果相对口腔健康儿童,重度龋齿患儿的牙菌斑微生物多样性增加,轻度龋齿患儿则减少,但变化不显著。门水平的优势菌主要为放线菌门、变形菌门及拟杆菌门,虽有相对丰度变化但差异无统计学意义。前10位的优势菌属中,轻度及重度龋齿儿童的最高丰度优势菌属为棒状杆菌属,重度龋齿儿童与健康儿童中的棒状杆菌丰度差异有统计学意义(t=-2.195 5, P=0.028 1)。健康儿童口腔的优势菌属包含了卟啉单胞菌属,但在轻度及重度龋齿儿童的优势菌属则替换为月形单胞菌属。种水平的菌群结构分析显示,前10位的优势菌丰度变化差异无统计学意义,而低丰度的致病菌如变异链球菌(H=27.302 1, P<0.000 1)、Atopobium parvulum(H=17.418 2, P=0.000 2)、栖牙普氏菌(H=10.598 9, P=0.005 0)、唾液普氏菌(H=10.035 0, P=0.006 6)等则在重度龋齿儿童中显著富集。结论低龄龋齿儿童的牙菌斑微生物结构发生了改变,部分口腔有害菌的丰度显著增加与龋病严重程度密切相关。