Salivary gel‐forming mucin MUC5B – a nutrient for dental plaque bacteria

Introduction: Model systems with oral bacteria from dental plaque have demonstrated that the utilization of complex glycoproteins as a food source cannot be undertaken by single species but requires concerted degradation by a multi‐species consortium, with each member contributing one or a few hydrolytic enzymes. Unlike previous studies, the aim of the present investigation was to explore the ability of fresh dental plaque to degrade salivary mucin, MUC5B, isolated by methods designed to retain intact the natural polymeric structure and physiological conformation, in an attempt to mimic the naturally occurring interaction between the oral microbiota and salivary mucins. Methods: Human salivary MUC5B was isolated from whole saliva by density‐gradient centrifugation and incubated with freshly isolated supragingival dental plaque with samples subjected to fluorescent staining for viability and metabolic activity. In addition, the degradation of MUC5B oligosaccharide side chains was studied using a lectin assay, recognizing three different carbohydrate epitopes commonly found on mucin oligosaccharide side chains. Results: The addition of purified salivary MUC5B elicited a strong metabolic response from the biofilm cells, whereas individual strains of Streptococcus oralis and Streptococcus gordonii isolated from the same plaque were not able to utilize the MUC5B. The degradation of terminal saccharide moieties on the MUC5B was demonstrated by a marked decrease in both sialic acid and fucose reactivity. Conclusion: These results have shown that dental plaque is capable of utilizing human salivary MUC5B as a nutrient source, a process possibly requiring the synergistic degradation of the molecule by a consortium of oral bacteria in the plaque community.     

牙菌斑生物膜的细菌生长抑制因子

牙菌斑生物膜中的细菌是龋病发生的先决条件,而口腔内存在多种细菌生长的抑制因子,它们对细菌的生长或菌斑的形成起一定抑制作用。细菌生长的抑制因子主要来自宿主和细菌本身产生的细菌素以及其他代谢产物,本文就致龋菌生长抑制因子的种类及作用的研究进展作一综述。     

Migration of leukocytes in dental pulp in response to plaque bacteria

Abstract – Cell material from three different dental plaque bacteria (Actinomyces viscosus., Streptococcus raitis, Actinobacillus actinomycetemcorriiitans) were studied for their capacity to induce leukocyte migration in the dental pulp of monkeys. Altogether five animals were used to provide 123 teeth for the study. The bacterial test materials were prepared from lyophilized sonicates of pure cultures of respective bacteria mixed with crystalline bovine serum albumin (BSA) 1:1 by weight. Immediately prior to use in the experiment saline was added to render the test material into a paste-like consistency. Buccal Class V cavities were prepared in the monkey teeth leaving a thin remaining wall to the pulp. In these cavities, the test materials were enclosed for a period of 8 h. A set of control cavities received BSA alone. Placement of test and control materials were made before and after depleting the animals of serum complement by repeated injections of Cobra Venom Factor. Histologic examination of pulp tissue specimens showed that materials from two of the bacterial species (A. viscosus, Act. actinomycetemcomitans) consistently induced infiltrations of polymorphonuclear leukocytes in one to several cell layers underneath the test cavity. The material from S. irtitis seemed less potent and in 11 of 22 teeth no pulpal reaction was recorded. Inacrivation of complement with Cobra Venom Factor did not appear to affect the severity of the pulpal responses.     

Comparison of five growth media and two anaerobic techniques for isolating bacteria from dental plaque

abstract — Five agar media in combination with the Hungate roll tube technique and conventional anaerobic jar technique (Baird & Tatlock(r) jars) were evaluated to determine the most suitable medium for non-selective isolation of the viable microorganisms in dental plaque. The highest colony count was obtained by using "Modified Medium 10" with the roll tube technique. About twice as many plaque colonies were recovered by roll tube technique as by the conventional anaerobic technique. With the MM 10 roll tube technique, 7 d of incubation revealed only 93% of the colonies that could be detected after 14 d of incubation.     

Streptococcus mutans-like bacteria from human dental plaque in a Sri Lanka (Ceylon) population

Abstract – This paper reports on the presence of low numbers of Streptococcus mutans among the oral streptococci present in human dental plaque in a Sri Lanka (Ceylon) population, where the caries activity is low and a low sucrose intake is combined with the presence of heavy plaque deposits. Plaque samples of unknown age were collected from 10 individuals in a tea estate, and another 10 samples were collected from dental students 19 days following interruption of oral hygiene. Of 670 such strains of oral streptococci studied, none showed typical "frosted glass" colony morphology on Mitis Salivarius agar. However, when subjected to physiological tests 14 of them were classified as S. mutans.     

Mucoepidermoid carcinoma-associated expression of MUC5AC,MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland

ObjectivesThe aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewis^a and sulfo-Lewis^a antigens) in MEC originating from the parotid gland, which normally does not secrete mucins.DesignWe conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included.ResultsWe observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewis^a and sulfo-Lewis^a were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland.ConclusionDuring the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered.     

A comparison of three methods for estimating dental plaque removal from individual teeth

The purpose of this study was to determine the efficiency of dental plaque removal, in vitro, by physical force. Of the 3 methods used, turbidity was the most sensitive for measuring microgram amounts of plaque. Air-dried weight and dental plaque imprint assays (DPIA) complemented turbidity measurements by providing a weight standard and a new method for visualizing plaque bacterial structure before the stained imprint was transformed into numerical data. The amount of dental plaque removed from a tooth by physical force can be measured and described as a percentage of the total removable plaque on that tooth.     

Lactoferrin, amylase and mucin MUC5B and their relation to the oral microflora in hyposalivation of different origins

There are several reasons for hyposalivation, each affecting the salivary composition in different ways. The aim of this study was to analyze and compare lactoferrin, amylase and mucin MUC5B in stimulated whole saliva collected from subjects with hyposalivation of different origins and to relate the results to the presence of some microbial species associated with oral disorders. Albumin was determined as a marker of serum leakage. The characteristic feature for subjects with radiation-induced hyposalivation was a large increase in lactoferrin, probably due to leakage through inflamed mucosal tissues, while it was a high albumin content for the group with primary Sj?gren's syndrome, probably due to disruption of the fragile mucosa. The saliva composition in subjects with hyposalivation of unknown origin or due to medicines was close to that in the healthy controls. All three hyposalivation groups tended to display a decrease in the concentrations of MUC5B and amylase. None of the microbial species analyzed (streptococci, mutans streptococci, Lactobacillus spp., Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Candida albicans, Staphylococcus aureus and enterics) correlated with concentration of MUC5B in saliva. The RT group, having the highest concentration of lactoferrin, had the lowest median number of F. nucleatum and was the only group in which median number of P. intermedia/P. nigrescens was zero.     

Serum antibodies to plaque bacteria in subjects with dental caries and gingivitis

abstract – Correlations were sought between indices of gingival inflammation and dental caries experience and serum antibody titers to five species of oral bacteria. The material comprised 53 young adult males. A statistically significant, negative correlation was observed between the antibody titer to a pool of Veillonella strains and dental caries experience. Multiple regression analyses failed to reveal significant associations between periodontal disease and serum antibody titers. However, the data suggested a combined association of the titers to the strains of Veillonella and a strain of Fusobacterium with the periodontal index.     

Effects of chlorhexidine on proteolytic and glycosidic enzyme activities of dental plaque bacteria

Chlorhexidine was tested for its ability to inhibit a wide range of glycosidic and proteolytic enzyme activities produced by Treponema denticola, Porphyromonas gingivalis, Bacteroides intermedius, Actinobacillus actinomycemcomitans, Capnocytophaga sputigena, Capnocytophaga gingivalis, Capnocytophaga orchracea, Capnocytophaga sp., Actinomyces viscosus, Streptococcus mitior, Streptococcus mutans, Streptococcus sobrinus, Streptococcus mitis, Streptococcus anginosus, Streptococcus oralis and Streptococcus sanguis. The enzymes produced by Capnocytophaga spp. were the most resistant to inhibition by chlorhexidine while the hydrolysis of proteolytic substrates by all the other species was markedly susceptible to inhibition with less than 0.125 mM chlorhexidine inhibiting enzyme activities by greater than or equal to 50%. Glycosidase activities, of all species, were generally more resistant to inhibition, especially neuraminidase activity. Chlorhexidine at less than 0.032 mM inhibited the degradation of bovine serum albumin by suspensions of dental plaque bacteria. These observations support an hypothesis that chlorhexidine exerts a bacteristatic effect in vivo, in part, by reducing the ability of dental plaque bacteria to degrade host-derived proteins and glycoproteins which normally provide essential nutrients for growth.     

Method for determination of invertase acitivity in homogentes of human dental plaque

Abstract – Supragingival human dental plaque was ecllected from patients with evidence of caries. The plaque was frozen and stored at-20°C. Pooled plaque was homogenized in acelate buffer pH 5.0 in an ice-water bath. By incubating the homogenate at pH 5.0 with [U-°C]-sucrose the formation fo glucose and fructose was followed. Incubation in acetate buffer at pH 5.0 eliminated the glycosyltransferase activities adn teh glycolytic pathway. Normal Michaclis-Menten kinetics were observed until about 40 mM sucrose. At highert concentration of sucrose, excess substrate inhibition occurred. Storage of the homogenate at −20° C resulted in decrease of the invertase activity with time.     

牙菌斑pH检测电极技术的研究进展

牙菌斑pH检测技术可检测龋风险,辅助预防龋病,其理论成熟、操作相对简单。由于临床龋风险检测技术的需求提高,微电极技术迅速发展,目前可实现牙菌斑pH检测的微电极种类也逐渐增加。现有电极技术主要包括微型玻璃电极、金属氧化物微电极以及离子敏感场效应晶体管。微型玻璃电极首先被应用于牙菌斑pH值的检测,但其结构薄弱,其中氧化铱微电极以其高强度、响应性优良的特点,成为近年来最具有应用潜力的接触法龋风险检测电极。金属氧化物微电极可以有效弥补玻璃电极强度不足的问题。随着电极技术进一步发展,更加小型、灵敏的离子敏感场效应晶体管受到研究者的关注。另外,为了克服接触法破坏菌斑结构的问题,近年来也有学者提出了光学测定技术,该技术可以实现无接触牙菌斑pH检测。未来的研究将在保证微小化的前提下进一步提高电极的强度及性能,并向无接触检测方向发展。     

Comparison of there anaerobic culture techniques and media for viable recovery of subgingival plaque bacteria

Abstract – Various methods are available for viable recovery of bacteria from subgingival plaque, but theire relative efficicency is not clear. In the first experiment, with 10 patients, three anaerobic techniques (Brewer jars, chamber and roll tubes) and three agar media (trypicase soy with 5% sheep blood (TS), brucella agar with 5% blood and 0.5 ug/ml of menadione (B), and modified medium 10 with 3% blood (MM 10)), were compared. In the second experiment, with anaother 10 patients, Brewer jars were compared with a glove box, using TS. Subgingival plaque was sampled with a gas flushed broach passed through a syringe. Significance of differences in viale recoveries was determined by ANOVA. Brewer jars and chamber (aerobic plating) were comparable in efficiency, but excelled roll tubes (anaerobic inoculation). TS was better than B, but equivalent to MM 10. TS in Brewer jars (aerobic plating) matched TS in the glove box (anaerobic plating), but perforemed better than all the other procedures tested. Recoveries did not differ on pre-and non-prereduced plates and on commercial and laboratory, freshly made platels. Non-evacuated jars sealed inside the box (anaerobic plating) provided lower recoveries than jars evacuated, filled and sealed outside (aerobic plating). Roll tubes benefited most by extending incubation from 7 to 14d. Commercial, non-prereduced TS agar plates incubated in Brewer jars or a glove box are highly recommendable methods for processing of subgingival plaque.     

Salivary factors and dental plaque levels in relation to the general health of elderly residents in a long‐term care facility: a pilot study

The goal of this study was to examine salivary factors and formation of dental plaque in elderly subjects who were institutionalized and to relate these factors to general health and medication use. This cross‐sectional study consisted of 50 elderly individuals who were institutionalized and 25 healthy young volunteers who served as a reference group. For each subject, salivary flow rates, salivary pH, and buffering capacity were assessed. Baseline Plaque Index and Plaque Formation Rate Index were used as a measure of oral hygiene. Information on each patient's general health and medication use was acquired from the institution. An evaluation of the results of this study suggests that salivary protective qualities and dental plaque levels in the elderly subjects who were institutionalized were worse than in healthy young individuals. An association between the general health of the elderly and salivary flow rate was found.     

Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque

The present study investigated a recently developed automated image analysis technique for its applicability to the enumeration of selected bacteria in supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples without user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, virtually indisruptable clumps with cells from multiple species. Due to their complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria was compared with visual counting of the fluorescence-labeled bacteria. Results are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oralis/Strep. sanguis, Strep. sobrinus, and Veillonella dispar/ V. parvula) with characteristic differences in abundance, cell morphology and aggregation behavior. Results revealed good correspondence between the two enumeration techniques (correlation coefficients ranging from 0.77 to 0.92) provided that the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully automated immunofluorescence to analyze such complex ecosystems as supragingival dental plaque.     

Application of stereological principles for quantification of bacteria in intact dental biofilms

Introduction:  Quantative confocal laser scanning microscopy (CLSM) in combination with fluorescent in situ hybridization (FISH) may help to increase our knowledge about biofilm formation. The purpose of this study was to develop and evaluate a stereological method for quantification of bacteria in intact biofilm. The method was applied in a quantitative study of the proportion of streptococci relative to other bacteria in initial in-situ -grown dental biofilms as a function of time.
Methods:  Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24, and 48 h. Biofilms were analysed using CLSM. Quantification of bacteria labelled with 16S ribosomal RNA oligonucleotide probes was performed with stereological tools: the unbiased counting frame and the two-dimensional fractionator.
Results:  Results showed a notable increase in the total number of bacteria and streptococci over time, with a considerable inter-individual variation at each time-point. After 48 h there was a 12.5-fold difference between individuals in the total number of bacteria and a 12.6-fold difference in the number of streptococci. The number of streptococci exceeded that of other bacteria and over the examination period there was a relatively constant relationship between the number of streptococci and other bacteria (streptococci vs. non-streptococci: median 15.2; minimum 1.0; maximum 89.3).
Conclusion:  The study demonstrates that the combined use of FISH and stereology is a relevant and reliable tool for obtaining unbiased information about the numerical contributions of specific bacterial populations during early biofilm formation.     

A digital SLR or intra-oral camera: preference for acquisition within an image analysis system for measurement of disclosed dental plaque area within clinical trials

Objectives:  The aim of this investigation was to compare the accuracy and reproducibility of images acquired from two types of camera (digital SLR camera, Kodak DCS410, KJP, UK and an intra-oral camera, Schick Technologies, UK) for application within an imaging system used for the quantification of disclosed dental plaque.
Methods:  Subjects refrained from brushing their teeth for 24 h. Their teeth were then disclosed with Erythrosin FDC Red 3 and duplicate sets of images were obtained by each examiner of the upper central and lateral incisors. Images were then saved and measured for dental plaque area. These data were used to calculate reliability.
Results:  No statistically significant bias in the measurements of plaque area was found. The reliability results showed the method was reliable. However, the image analysis system incorporating the 35 mm SRL camera was more reliable for both operators, as demonstrated by their intra-operator results. This was also collaborated by the inter-operator results.
Conclusion:  In this investigation the digital SLR camera combined with the image analysis system and frame permitted greater reliability of dental plaque surface area measurements than the digital intra-oral camera. This conclusion was derived both from the reliability data and from the perceived ease of use and flexibility of both camera types. When combined with the use of the custom-made frame, the images using the digital SLR camera were highly reproducible, confirming the systems application within clinical trials.     

Risk for the development of atherosclerosis in women with a high amount [corrected] of dental plaque and severe gingival inflammation

Abstract Introduction: Chronic infection and inflammation are considered to be risk factors in the development of cardiovascular diseases; the chronic inflammatory and microbial burden caused by the dental plaque in these individuals may predispose them to atherosclerotic process. Aims: The aims were to study the involvement of a high level of dental plaque, severe gingival inflammation and periodontitis in the development of early atherosclerotic process in women. Methods: Forty‐six randomly chosen women with periodontitis and 21 periodontally healthy women were subjected to a comprehensive clinical oral examination, including oral hygiene status and level of gingival inflammation. Atherosclerotic risk factor analysis and carotid ultrasonography were performed. Common carotid artery intima‐media thickness (IMT) and lumen diameter were measured and intima‐media area (cIMA) was calculated. The following statistical methods were used: analysis of variance, chi‐squared tests and multiple logistic regression analysis. Results: There were highly significant differences between the patients and controls in the amount of dental plaque, gingival inflammation as well as bleeding on probing and pocket depth. The mean values of IMT and cIMA were significantly higher in women with periodontal disease than in controls. Multiple logistic regression analysis identified periodontitis as a principal‐independent predictor of both the common carotid artery cIMA and IMT. Conclusions: The present results indicate that a high amount of dental plaque, severe gingival inflammation as well as periodontitis seem to be associated with the development of atherosclerotic lesions in women already at its early and subclinical stages.