Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00506.x Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers Objective: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. Materials and methods: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher’s exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. Results: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue‐coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. Conclusion: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.     

Inhibitory effect of galectin-3 on the cytokine-inducing activity of periodontopathic Aggregatibacter actinomycetemcomitans endotoxin in splenocytes derived from mice

Galectins, a family of animal lectins, are involved not only in development and differentiation but also in immunoregulation and host–pathogen interactions. Galectin-3 interacts with lipopolysaccharides in gram-negative bacteria such as Escherichia coli, Salmonella minnesota and Pseudomonas aeruginosa . The present study investigated whether galectin-3 inhibited the cytokine-inducing activity of periodontopathic bacterial lipopolysaccharides using splenocytes derived from mice of different ages. Lipopolysaccharides were extracted from Aggregatibacter actinomycetemcomitans Y4 and Porphyromonas gingivalis ATCC 33277, and then purified. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that galectin-3 adhered to A. actinomycetemcomitans lipopolysaccharides, but not to the lipopolysaccharides of P. gingivalis . Splenocytes were prepared from 1- or 7-month-old C57BL/6 mice. Either A. actinomycetemcomitans lipopolysaccharides (200 ng mL⁻¹) alone or lipopolysaccharides and murine galectin-3 (10 μg mL⁻¹) were added to culture solutions, and the release of interleukin-6 (IL-6) and interferon-γ (IFNγ) from splenocytes was measured by ELISA after a 17-h incubation. In all mice tested, A. actinomycetemcomitans lipopolysaccharide stimulation significantly increased the production of IL-6 and IFNγ ( P <0.01). Murine galectin-3 suppressed lipopolysaccharide-induced cytokine production in the splenocytes of the 1-month-old mice ( P <0.02 for IL-6; P <0.05 for IFNγ), but not in the splenocytes of the 7-month-old mice. This suggests that responses change with age.     

Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR

Aims:  Breast milk has been described as a source of bacteria influencing the development of the infant gut microbiota. Up to the present, few studies have been focused on the application of culture-independent techniques to study bacterial diversity in breast milk. In this context, the aim of this study was to characterize the breast milk microbiota of healthy women by applying the quantitative real-time PCR technique (qRTi-PCR).
Methods and Results:  A total of 50 breast milk samples were analysed by qPCR to assess the presence of different bacterial genera or clusters, including the Bifidobacterium , Lactobacillus , Staphylococcus , Bacteroides , Enterococcus , Streptococcus , Clostridium cluster IV and Clostridium cluster XIVa–XIVb groups. Staphylococcus , Streptococcus , Bifidobacterium and Lactobacillus were the predominant groups and were detected in all the samples. Clostridium XIVa–XIVb and Enterococcus were detected in most of the samples in contrast to the Bacteroides and Clostridium cluster IV groups.
Conclusions:  Our results confirm the abundance of bacterial DNA in breast milk samples and suggest that the qRTi-PCR technique has a huge potential in the microbiological analysis of human milk.
Significance and Impact of the study:  qRTi-PCR allowed the detection of bacterial DNA of streptococci, staphylococci, lactic acid bacteria and bifidobacteria in the samples of human milk, which confirms that breast milk can be an important source of bacteria and bacterial DNA to the infant gut.     

复合探针荧光定量PCR法检测布鲁氏菌的实验研究

目的：建立用复合探针荧光定量PCR快速检测布鲁氏菌的方法。方法：研究根据BSCP31基因编码31KDa的布鲁氏杆菌表面蛋白的核苷酸序列设计特异引物,通过PCR法的特异性、灵敏度和重复性研究,建立了复合探针荧光定量PCR检测布鲁氏菌的方法。用于布鲁氏菌病的筛选和诊断。结果：结果表明该检测方法的特异性为100％,最低可检出10个拷贝的质粒DNA分子,可对1×10^1-1×10^6拷贝范围内的模板进行定量,最低可检测至1×10^2CFU／ml细菌。该方法的精密度好,阳性质控品和阴性质控品不同时间测定三次及同一时间五次重复实验结果CV值均小于5％。结论：本研究建立的复合探针实时荧光定量PCR检测布鲁氏杆菌的方法,可对布鲁氏病原菌进行快速检测,对布病的筛选和确诊具有重要意义。     

Nicotine involved in periodontal disease through influence on cytokine levels

Periodontal disease, for which smoking is a known risk factor, is infectious, and is associated with oral biofilm. Cytokines mediate and regulate immune and inflammatory responses. Lipopolysaccharide produced by periodontopathic bacteria plays a role in the progression of periodontitis. The effect of nicotine on cytokine production in mice was evaluated in this study. Nicotine (10 or 200 microg mouse(-1)) was administered intraperitoneally to 4-week-old female BALB/c mice, once a day, for 49 days. Control mice received injections of phosphate-buffered saline. Blood was collected from all mice and serum IL-6, IL-10, tumor necrosis factor (TNF)-alpha and IFN-gamma levels were measured by an enzyme-linked immunosorbent assay on the 42nd day. IL-6, IL-10 and IFN-gamma levels in the nicotine-treated mice were higher than those in the control mice. However, no differences were found in TNF-alpha levels between nicotine-treated and control mice. Lipopolysaccharide (20 microg mouse(-1)) purified from Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans) Y4 was administered intraperitoneally on the 49th day. A rapid increase in TNF-alpha was observed in the control mice at 2 h after administration of lipopolysaccharide. In contrast, no increase was noted in the nicotine-treated groups. Significantly higher levels of IFN-gamma were seen in the 200 microg nicotine-treated mice at 2 h after administration of lipopolysaccharide (P<0.05). The results showed that cytokine levels were influenced by nicotine in mice.     

复合探针荧光定量PCR法检测布鲁氏菌的实验研究

目的:建立用复合探针荧光定量PCR快速检测布鲁氏菌的方法。方法:研究根据BSCP31基因编码31KDa的布鲁氏杆菌表面蛋白的核苷酸序列设计特异引物,通过PCR法的特异性、灵敏度和重复性研究,建立了复合探针荧光定量PCR检测布鲁氏菌的方法,用于布鲁氏菌病的筛选和诊断。结果:结果表明该检测方法的特异性为100%,最低可检出10个拷贝的质粒DNA分子,可对1×101-1×106拷贝范围内的模板进行定量,最低可检测至1×102CFU/ml细菌。该方法的精密度好,阳性质控品和阴性质控品不同时间测定三次及同一时间五次重复实验结果CV值均小于5%。结论:本研究建立的复合探针实时荧光定量PCR检测布鲁氏杆菌的方法,可对布鲁氏病原菌进行快速检测,对布病的筛选和确诊具有重要意义。     

Changes of immunoresponse in BALB/c mice neonatally treated with periodontopathic bacterial endotoxin

The aim of this investigation was to analyze the effects of early life exposure to periodontopathic bacterial lipopolysaccharides on immunoresponse. Newborn BALB/c mice were subcutaneously injected with 20 ng lipopolysaccharide of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans or Escherichia coli daily for 2 days, starting within 24 h after birth. The treated mice were given intraperitoneal injections of bovine serum albumin at 180 and 187 days of age. Seventeen hours after each injection, the mice were bled and sera were separated. Their sera were tested in an enzyme-linked immunosorbent assay system. The mean interleukin-4, interleukin-5, interleukin-6 and immunoglobulin E levels in the sera of mice treated neonatally with P. gingivalis lipopolysaccharide were significantly higher than those of the controls. However, in all cases, no significant difference was noted between mice treated neonatally with A. actinomycetemcomitans- or E. coli lipopolysaccharide and control mice. These data suggest that neonatal exposure to P. gingivalis lipopolysaccharide induces changes in immunological responses when the mice reach maturity.     

Prevalence of periodontopathic bacteria on the tongue dorsum of elderly people

Objectives: The aim of this study was to analyse the prevalence of oral bacteria on the dorsum of the tongue. In addition, the relationship between the number of teeth and the microflora present on the coating of the tongue in a population of 85‐year‐old people was assessed. Subjects and methods: Two hundred and five individuals (89 males, 116 females) from the same geographical area who were 85 years of age were examined. Five periodontopathic bacteria (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Treponema denticola) and one cariogenic bacterium (Streptococcus mutans) were analysed using a polymerase chain reaction assay of tongue samples from the population. Results: Periodontal bacteria‐positive individuals have more teeth than that of periodontal bacteria‐negative people. Between the periodontal bacteria‐positive and ‐negative individuals, there were significant differences in the mean number of teeth for P. gingivalis (p < 0.0001), T. denticola (p < 0.001), F. nucleatum (p = 0.002), and T. forsythia (p = 0.005), while there were no significant differences for A. actinomycetemcomitans (p = 0.998) or S. mutans (p = 0.147). Conclusions: A wide range of species, including anaerobes, was detected in 85‐year‐old subjects. It was found that the detection of periodontal bacteria on the tongue coating increased with the number of teeth. There was a positive relationship between the tooth number and periodontopathic bacteria, except for A. actinomycetemcomitans.These results suggest that tongue care is essential for preventing oral disease and needs to be part of any oral care programme in elderly people.     

基于实时定量PCR技术的育龄期细菌性阴道病患者阴道菌群结构分析及其临床应用

目的 探讨育龄期女性细菌性阴道病（bacterial vaginosis,BV）患者阴道优势菌群变化及其与临床指标的相关性。方法 选择本院门诊确诊的育龄期BV患者35例和同期入院体检的健康体检女性37例,无菌拭子采集阴道中段壁分泌物,提取细菌基因组DNA,采用实时定量PCR技术（real-time quantitative PCR,qPCR）进行阴道优势细菌检测,并进行其与临床指标如阴道pH和Nugent评分相关性分析。结果 乳杆菌属细菌及其种水平的细菌如惰性乳杆菌、卷曲乳杆菌、詹氏乳杆菌等在BV患者中均显著下降,BV相关阴道致病细菌如加德纳菌属、奇异菌属、埃格特菌属、巨型球菌I型菌属、纤毛菌属和普氏菌属显著升高（P<0.05）。阴道致病菌群与阴道pH和Nugent评分呈显著正相关,而阴道卷曲乳杆菌和惰性乳杆菌与其呈负相关。结论 育龄期BV患者阴道优势菌群显著失衡,并与阴道pH和Nugent评分显著相关,提示阴道优势菌群改变参与BV发生发展。     

新定量PCR数据处理方法的理论探讨

日新月异的生命科学技术的发展及临床医学科学研究的需求,一般的PCR技术已远远不能满足工作的需要。PE公司在进行了大量的PCR动力学研究的基础上,发现了利用荧光标记探针在PCR循环过程中积累的荧光强度达到仪器捡出阈值时,系统的初始模板数量与循环次数之间有线性关系,据此建立了目前的PE 7700 、PE 5700仪器的定量PCR技术,开创了PCR技术的新局面。但是由于这一技术的误差较大,尚不能满足生命科学及临床医学科学研究的需求,因此需要继续研究新的定量PCR技术。PCR动力学数学模型是根据PCR 技术的原理提出的,能够准确描述PCR反应产物分子数量积累规律的动力学方程,给出了PCR产物数量或者荧光强度与初始模板数量及其他反应条件间的函数关系。利用这一关系,根据PCR反应已积累的产物数量,可以实现准确的定量PCR分析,得到初始模板数量达到定量PCR的目的。使用动力学数学模型做定量PCR分析,其结果的误差仅与使用的荧光强度数值的精确度相关。使用精确到6位数的荧光强度数据,模板数自100~1 000 000区间定量结果的准确性可达99%以上。本文根据模拟实验数据进行了初步的定量PCR分析,结果提示,目前的定量PCR仪器使用PCR动力学模型理论处理分析数据,定量分析的结果会比目前的CT值方法在准确性方面提高几十倍以上,可以满足各方面研究工作误差水平的需要。Abstract:Today standard PCR can't satisfy the need of biotechnique development and clinical research any more.After numerous dynamic research,PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700.But the error of this technique is too great to satisfy the need of biotechnique development and clinical research.A better quantitative PCR technique is needed.The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system.This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions,and can reflect the accumulating rule of PCR product molecule accurately.Accurate quantitative PCR analysis can be made use this function relation.Accumulated PCR product quantity can be obtained from initial template number.Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used.For an example,when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1 000 000,the quantitative result accuracy will be more than 99%.The difference of result error is distinct using same condition,same instrument but different analysis method.Moreover,if the PCR quantitative analysis system is used to process data,it will get result 80 times of accuracy than using CT method.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

Periodontopathic bacterial endotoxin-induced tumor necrosis factor alpha production was inhibited by exercise in mice

The effects of exercise on serum interleukin-6 and tumor necrosis factor alpha levels were investigated using mice. Five-week-old female BALB/c mice (Th2-biased) and C57BL/6 mice (Th1-biased) were divided into exercise and control groups. The exercise group was exercised in a rotating basket type treadmill for 1 h (5 r.p.m.). Blood was collected and the serum was separated immediately after exercise. The serum interleukin-6 and tumor necrosis factor alpha levels were measured using an Endogen ELISA kit. Exercise significantly increased the serum interleukin-6 level in the two strains of mice (P<0.05 and P<0.01). The tumor necrosis factor alpha level was decreased in the exercise group. Next, periodontopathic bacterial endotoxin (lipopolysaccharide) was administered after exercise, and the effects of exercise on the lipopolysaccharide-induced serum interleukin-6 and tumor necrosis factor alpha levels were investigated. Exercise inhibited lipopolysaccharide-induced tumor necrosis factor alpha production, suggesting it has a defensive action against endotoxin shock.     

牙周可疑致病菌与下呼吸道感染的关系

牙菌斑生物膜是牙周炎的始动因子。研究发现,牙周炎与全身多器官或系统的感染性疾病有关,如心脑血管疾病、糖尿病、消化道和呼吸道疾病。近年来,牙周炎与呼吸道疾病之间的关系备受关注,本文将近期对牙周可疑致病菌感染下呼吸道的途径及其作用研究做简要综述。     

Novel primers for detection and quantification of Myxococcus species in situ

A nested polymerase chain reaction (PCR) protocol using unique primers was developed to detect and quantify Myxococcus species from environmental samples. The protocol amplified most species of Myxococcus when 10 pg of DNA representing 1000 cells was present, although over half were amplified with as little as 0.1 pg (10 cells). The protocol did not amplify other myxobacterial species, members of the δ‐proteobacteria or other unrelated organisms tested at significantly higher concentrations of DNA. The primers were also used in quantitative PCRs, which accurately estimated the population levels in soil.     

乙型肝炎患者HBV复制水平的定量PCR研究

本文采用定量ＰＣＲ方法观察了慢性乙型肝炎患者ＨＢＶ－ＤＮＡ的复制水平。结果表明在ＨＢｅＡｇ阳性患者血清ＨＢＶ－ＤＮＡ检出率和水平显著高于ＨＢｅＡｇ阴性患者（Ｐ＜０．０１）;血清ＨＢＶ－ＤＮＡ水平的高低与ＡＬＴ异常情况未发现有相关性。