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.     

Plaque formation in vitro by Actinomyces viscosus in the presence of Streptococcus sanguis or Streptococcus mutans.

Actinomyces viscosus, growing on a tooth in the presence of sucrose, slowly produced a loosely-attached plaque, the pH being 6.1 after 120 h. When the tooth was inoculated simultaneously with A. viscosus and either Streptococcus sanguis or Streptococcus mutans, firmly-adherent plaque was quickly formed and the pH fell below 5 after 33 h with the former Streptococcus and 24 h with the latter. A. viscosus disappeared from each mixed plaque by 120 h.     

Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.     

白及提取物抗变异链球菌致龋效果的研究

目的研究白及提取物对变异链球菌粘附、生物膜形成及活性的影响,评价其抗龋效果。方法市售白及95%乙醇浸提;纸片法、打孔法测定直接抑菌作用;液体稀释法检测MIC;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用荧光显微镜和激光共聚焦显微镜观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响;运用扫描电镜观察白及药液对变异链球菌生物膜的影响。结果白及提取物具有一定的抑菌作用,MIC为16~62 mg/m L;结晶紫法定量研究生物膜结果显示白及药液作用4 h对变异链球菌的粘附均有抑制作用,抑制率为28.63%~60.08%;作用20 h对生物膜总量抑制率达77.08%;白及药液作用20 h,荧光染色显示生物膜活性明显被抑制,抑制率达62.03%;梯度浓度白及药液分别作用20 h后,激光共聚焦显微镜下观察到随着药液浓度增加,绿色的活菌、团块状结构减少,生物膜形成明显被抑制;扫描电镜下可见药液作用后细菌间粘性物质减少。结论高浓度白及提取液对变异链球菌有直接抑菌作用,亚抑菌浓度能抑制其粘附和生物膜的形成,进而具有抗龋作用。     

Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159

Streptococcus mutans has at least six pairs of open reading frames that are homologous to bacterial two-component regulatory systems. Putative response regulators from five out of six of these pairs were successfully mutated by insertion of a kanamycin resistance marker and the effects of inactivation of the genes on the ability of the cells to form biofilms in an in vitro model were assessed. Disruption of the response regulators of four systems had no effect on biofilm formation, whereas disruption of one response regulator caused a substantial decrease in biofilm formation as compared to the wild-type S. mutans.     

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。     

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.     

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.     

Lack of in vitro biofilm formation does not attenuate the virulence of Streptococcus gordonii in experimental endocarditis

The ability to induce experimental endocarditis of biofilm-deficient mutants of Streptococcus gordonii was studied in an isogenic background. Strains were inactivated in either comD, fruK or pbp2b genes, which are involved in biofilm formation. These strains were clearly impaired (>75% reduction) in biofilm production in vitro. However, this did not result in a decreased severity of infection in vivo.     

Identification of the Streptococcus gordonii glmM gene encoding phosphoglucosamine mutase and its role in bacterial cell morphology, biofilm formation, and sensitivity to antibiotics

Phosphoglucosamine mutase (EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. The gene (glmM) of Escherichia coli encoding the enzyme has been identified previously. We have now identified a glmM homolog in Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and have confirmed that the gene encodes phosphoglucosamine mutase by assaying the enzymatic activity of the recombinant GlmM protein. Insertional glmM mutant of S. gordonii did not produce GlmM, and had a growth rate that was approximately half that of the wild type. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, and increased roughness of the bacterial cell surface. Furthermore, the glmM mutation reduces biofilm formation and increases sensitivity to penicillins relative to wild type. All of these phenotypic changes were also observed in a glmM deletion mutant, and were restored by the complementation with plasmid-borne glmM. These results suggest that, in S. gordonii, mutations in glmM appear to influence bacterial cell growth and morphology, biofilm formation, and sensitivity to penicillins.     

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

Abstract

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p?<?0.05). Hot steeping significantly reduced bacterial growth (p?<?0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.     

Surface properties,adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans

Abstract

In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4?h of fungi–surface contact. Additionally, after 72?h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72?h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.     

Effect of honey on Streptococcus mutans growth and biofilm formation

Because of the tradition of using honey as an antimicrobial medicament, we investigated the effect of natural honey (NH) on Streptococcus mutans growth, viability, and biofilm formation compared to that of an artificial honey (AH). AH contained the sugars at the concentrations reported for NH. NH and AH concentrations were obtained by serial dilution with tryptic soy broth (TSB). Several concentrations of NH and AH were tested for inhibition of bacterial growth, viability, and biofilm formation after inoculation with S. mutans UA159 in 96-well microtiter plates to obtain absorbance and CFU values. Overall, NH supported significantly less (P < 0.05) bacterial growth than AH at 25 and 12.5% concentrations. At 50 and 25% concentrations, both honey groups provided significantly less bacterial growth and biofilm formation than the TSB control. For bacterial viability, the results for all honey concentrations except 50% NH were not significantly different from those for the TSB control. NH was able to decrease the maximum velocity of S. mutans growth compared to AH. In summary, NH demonstrated more inhibition of bacterial growth, viability, and biofilm formation than AH. This study highlights the potential antibacterial properties of NH and could suggest that the antimicrobial mechanism of NH is not solely due to its high sugar content.     

Probiotic Lactobacillus sp. inhibit growth,biofilm formation and gene expression of caries‐inducing Streptococcus mutans

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real‐time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH‐neutralized, catalase‐treated or trypsin‐treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH‐dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide‐dependent antimicrobial activities. All biofilm‐forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.     

Predominance of non-Streptococcus mutans bacteria in dental biofilm and its relation to caries progression

This study aims to assess differences in biofilm bacterial composition between patients with low and high caries. Patients without a medical problem and with no history of antibiotic use, mouth wash or fluoride application in the previous 3 months were recruited. Caries was recorded at cavitation level; score was calculated by a national mean (dmft of 4.8 and DMFT of 2.7). Pooled biofilm samples were collected from mesial, distal, buccal, lingual, and occlusal surfaces. Based on caries experience, individuals were classified into low and high caries and both groups were compared regarding bacteria identified using 16S rRNA gene sequencing, and molecular phylogenetic analysis of the isolates was performed. A total of twenty seven randomly selected samples with low (n = 13) and high (n = 14) caries. Identification of oral bacteria was performed using 16S rRNA sequence, Rothia mucilaginosa and R. aeria were identified in low caries individuals, while R. dentocariosa was detected in high caries individuals. Two Streptococcus spp. were identified only in low caries S. salivarius and S. gordonii whereas S. sanguinis, S. mitis, S. sinensis, S. rubneri, S. vestibularis, S. cristatus and S. massiliensis were identified only in individuals with high caries. This study revealed the absence of R. mucilaginosa in the high caries subjects and its coexistence with the low caries subjects. Streptococcus mutans was insignificant contributor of caries among samples, while, Streptococcus sanguinis was the main constituent of high caries Saudi patients.     

Growth of mixed cultures of Actinomyces viscosus and Streptococcus mutans under dual limitation of glucose and oxygen

Abstract Streptococcus mutans and Actinomyces viscosus are among the dominant species in human dental plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to occur frequently. Therfore, mixed cultures of the 2 species were studied under dual limitation of glucose and oxygen. Over a wide range of oxygen-supply rates, coexistence of A. viscosus and S. mutans was observed, within this range A. viscosus increased almost linearly with oxygen supply. A mathematical model based on Monod-type type kinetics and accounting for uncompetitive inhibition of growth by oxygen was developed to simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the results of the chemostat experiments, led to the conclusion that coexistence of the two species     

Protein expression by Streptococcus mutans during initial stage of biofilm formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.