Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.     

Enhancing CCL28 expression through the gene transfer to salivary glands for controlling cariogenic microbe

The chemokine CCL28 participates in direct antimicrobial activities as well as homing of certain types of lymphocytes. The present study was conducted to harness these properties of the chemokine for the prevention of dental caries. The gene encoding CCL28 was transferred to salivary glands to enhance the production of this chemokine locally. First, a recombinant eukaryotic plasmid expressing CCL28 was constructed. Then, the CCL28 protein from 293 cells transfected with the recombinant plasmid was verified to inhibit the caries pathogen Streptococcus mutans (S. mutans) in a biofilm. Finally, the recombinant plasmid was retrogradely administered to the parotid glands of rats through the secretory ducts. The successful transfer of the gene encoding CCL28 to rat parotid acinar cells was confirmed by immunofluorescence and real-time PCR. Increases in both CCL28 and secretory IgA (SIgA) in the rat saliva were tested by ELISA. It was revealed that the CCL28 protein obtained from the study was able to strongly inhibit S. mutans living in biofilm in vitro. The delivery of the recombinant plasmid to the rat parotid glands was able to induce high levels of CCL28 and SIgA in saliva, and the increased levels of CCL28 and SIgA in saliva were maintained for 2 weeks. Notably, the dental plaque from the rats treated with the delivery of the recombinant plasmid in the study harbored significantly less S. mutans. These data indicated that the present strategy may hold hope for the effective prevention of dental caries.     

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model

BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

Elevated incidence of dental caries in a mouse model of cystic fibrosis

Background

Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model.

Methodology/Principal Findings

We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value<0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16±2 mM vs. 31±2 mM, CF and wildtype mice, respectively; p value<0.01; t test).

Conclusions/Significance

Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.     

The anti-biofouling effect of Lactobacillus fermentum-derived biosurfactant against Streptococcus mutans

Biofouling in the oral cavity often causes serious problems. The ability of Streptococcus mutans to synthesize extracellular glucans from sucrose using glucosyltransferases (gtfs) is vital for the initiation and progression of dental caries. Recently, it was demonstrated that some biological compounds, such as secondary metabolites of probiotic bacteria, have an anti-biofouling effect. In this study, S. mutans was investigated for the anti-biofouling effect of Lactobacillus fermentum (L.f.)-derived biosurfactant. It was hypothesized that two enzymes produced by S. mutans, glucosyltransferases B and C, would be inhibited by the L.f.-biosurfactant. When these two enzymes were inhibited, fewer biofilms (or none) were formed. RNA was extracted from a 48-h biofilm of S. mutans formed in the presence or absence of L.f. biosurfactant, and the gene expression level of gtfB/C was quantified using the real-time polymerase chain reaction (RT-PCR). L.f. biosurfactant showed substantial anti-biofouling activity because it reduced the process of attachment and biofilm production and also showed a reduction in gtfB/C gene expression (P value?相似文献   

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.     

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.     

变形链球菌中的次级代谢产物及其在口腔生物被膜中的生态功能

口腔生物被膜是由数百种微生物构成的复杂微生物群体。变形链球菌作为其中的重要一员,被认为是引起龋病的主要病原菌。变形链球菌在牙齿表面以生物被膜形式生长的能力和它产酸耐酸的特点赋予其致龋性。不同的变形链球菌菌株之间保留了多样的次级代谢形式,这是与宿主长期共进化的结果。迄今为止,变形链球菌中报道的次级代谢产物包括10种细菌素(又称变链素)和一种聚酮/非核糖体肽类化合物。这些化合物多样的活性形式暗示了它们参与口腔生物被膜中跨种间和跨界间的相互作用。未来随着变形链球菌菌株数目的增加和更多菌株全基因组序列的完成,可以预见会有更多新颖活性次级代谢产物的出现。对变形链球菌次级代谢的研究不仅有助于治疗和预防口腔疾病,而且新颖活性次级代谢产物的发现对新药的研发也具有重要意义。     

变形链球菌对不同牙科充填材料的粘附及早期生物膜的形成

目的探讨变形链球菌对不同牙科充填材料的粘附和早期生物膜的形成.方法比较经放射性同位素3H-TDR(3H-胸腺嘧啶核苷)标记的变形链球菌对3种唾液包被的充填材料的粘附.采用蛋白质测量试剂盒定量分析其对唾液蛋白的吸附量;采用凝胶电泳和图像分析系统定量分析其对唾液白蛋白和α-淀粉酶的吸收率.结果各种材料对变形链球菌的粘附能力,对唾液蛋白的吸附能力均随着材料的不同而不同.Fuji IX对细菌的粘附量很高,但是对蛋白的吸附量却很低;而F2000对细菌的粘附量很低,对蛋白的吸附量却很高.结论在不同充填材料表面形成的生物膜是不同的,提示早期生物膜的形成具有一定的特异性.这种生物膜的差异对口腔微生态环境及龋病和/或牙周病的发展具有重要意义.     

A whole cell BIAcore assay to evaluate P1-mediated adherence of Streptococcus mutans to human salivary agglutinin and inhibition by specific antibodies

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.     

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.     

Effect of mutacin administration on Streptococcus mutans-induced dental caries in rats

A bacteriocin from serotype c Streptococcus mutans strain C3603 was examined for its inhibitory effect on experimental dental caries in rats infected with S. mutans MT8148R (serotype c). Significant reduction in the incidence of dental caries was found only when bacteriocin was incorporated both in the drinking water and in the diet at a high concentration. However, caries reduction was not as great as expected and the addition of bacteriocin to drinking water alone had no effect on the recovery of S. mutans, plaque deposition or caries incidence. The bacteriocin activity must have been reduced in the oral cavity of rats, and the reasons were examined. Bacteriocin-resistant mutants were not detected and the bacteriocin was not inactivated by saliva. Whereas the bacteriocin did not kill the S. mutans cells grown in a sucrose-containing medium, it completely killed the cells grown in a sucrose-free medium.     

Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen

Streptococcus mutans is known to be a major causative organism of human dental caries. A surface protein Ag with a molecular mass of 190 kDa of S. mutans (PAc) is receiving attention as an anticaries vaccine. We have recently determined the complete nucleotide sequence of the gene for PAc. In this study, four peptides were synthesized on the basis of amino acid sequence of PAc. Among these peptides, PAc(301-319) corresponding to the alanine-rich repeating amino acid region was the most strongly bound by polyclonal murine anti-rPAc antibodies. The peptide partially inhibited the binding of polyclonal anti-rPAc antibodies to rPAc. The peptide induced the proliferation of T cells from BALB/c mice immunized with rPAc. Subcutaneous immunization with PAc(301-319) or rPAc emulsified in CFA/IFA induced high serum IgG responses to rPAc and PAc(301-319). In addition, serum IgG responses to a surface protein Ag with a molecular mass of 210 kDa of Streptococcus sobrinus were elicited in mice immunized by s.c. injection with PAc(301-319) or rPAc. Intranasal immunization with PAc(301-319) coupled to cholera toxin B subunit (CTB) or with rPAc and free CTB induced high serum IgG responses to rPAc. The immunization with PAc(301-319) coupled to CTB or rPAc and free CTB suppressed the colonization of murine teeth by S. mutans. These results suggest that intranasal immunization with the peptide or rPAc may be effective for the prevention of dental caries.     

Differences in microbiological composition of saliva and dental plaque in subjects with different drinking habits

Several foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dental crown caries, as well as inhibition of glucosyltransferases, which are the S. mutans enzymes involved in the synthesis of an adherent, water-insoluble glucan from sucrose. Other studies have demonstrated an in vitro action on oral plaque biofilm formation and desorption. This study evaluated whether the activity displayed in vitro by food compounds could affect the microbiological composition of saliva and dental plaque of subjects with a diet rich in these foods, comparing the results with those obtained from subjects with a different diet. The foods considered were: coffee, barley coffee, tea and wine. A total of 93 subjects were recruited into the study. Six samples of both plaque and saliva were collected from each subject at roughly one-monthly intervals. Total bacteria, total streptococci, S. mutans and lactobacilli counts were determined by culture in both saliva and dental plaque. The highest bacterial titres were recorded for the control population, while each drinking habit subgroup showed counts roughly one log lower than the controls. These differences in bacterial counts proved statistically significant (P<0.05). As far as dental plaque was concerned, while total counts did not significantly vary per mg of plaque in the subjects belonging to the different drinking habit subgroups, a significant decrease (P<0.05) was observed in those subjects drinking coffee, tea, barley coffee and wine when mutans streptococci and lactobacilli were evaluated. In several cases a more than one log decrease was observed. Plaque indices were also determined, and a significant (P<0.05) reduction in values was recorded in the subjects belonging the specific drinking habit subgroups compared to the control group. This study indicates that there is a correlation between consumption of specific foods and oral health in terms of reduced plaque deposition and lower counts of odontopathogens.     

Streptococcus mutans and oral streptococci in dental plaque

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.