Effect of the antimicrobial decapeptide KSL on the growth of oral pathogens and Streptococcus mutans biofilm

Dental caries and periodontitis are common oral bacterial infectious diseases. Their prevention and treatment requires control of the causative pathogens, such as Streptococcus mutans and Porphyromonas gingivalis, that exist within dental plaque. As one of the attractive future substitutes for conventional antibiotics, antimicrobial peptides (AMPs), both natural and synthetic, have been widely tested and used for controlling bacterial infections. In this study, we investigated the antimicrobial activity of KSL (KKVVFKVKFK-NH(2)), a novel AMP, against several major cariogenic and periodontopathogenic bacteria as well as Candida albicans in vitro. Streptococcus mutans, the causative agent of dental caries, was chosen for in-depth testing. Bacterial susceptibility and time-kill assays were performed to investigate the sensitivity of S. mutans to KSL. The effect of KSL on biofilm formation and on pre-formed biofilm was also examined. For biofilm studies, confocal laser scanning microscopy (CLSM) was used to observe and analyse bacterial biofilm. The results showed that KSL had antimicrobial activity against a variety of oral bacteria and fungi. Streptococcus mutans and Lactobacillus acidophilus were the most susceptible strains to KSL peptide [minimum inhibitory concentration (MIC) of 0.0625 mg/mL] compared with other species tested (MICs of 0.125-1mg/mL). KSL also inhibited S. mutans biofilm formation, with a minimum biofilm inhibition concentration of 0.0625-0.125 mg/mL, and reduced 1-day-old developed S. mutans biofilm, with a minimum biofilm reduction concentration of 0.25-0.5mg/mL. CLSM images showed that KSL significantly reduced the viability of biofilm cells. This study suggests that KSL may have a potential clinical application in treating dental caries by killing S. mutans within dental plaque.     

Antimicrobial and anti-plaque activity of N'-alkyl-N-(2-aminoethyl)piperidine against dental plaque bacteria

Five N'-alkyl-N-(2-aminoethyl)piperidines were synthesized and their in vitro antimicrobial activities were tested against four micro-organisms related to dental caries (Streptococcus mutans, S. sobrinus, Actinomyces viscosus, and A. naeslundii) which are known to be implicated in dental caries. The tetradecyl and hexadecyl derivatives possessed good bacteriostatic activity. Some derivatives exhibited a rapid bactericidal effect against S. mutans and S. sobrinus in aqueous solution. These compounds also possessed surfactant properties and anti-plaque activity.     

Isopanduratin A from Kaempferia pandurata as an active antibacterial agent against cariogenic Streptococcus mutans

An antibacterial compound active against Streptococcus mutans was isolated from Kaempferia pandurata and identified as isopanduratin A using 1H NMR, 13C NMR and EI-MS. The minimum inhibitory concentration (MIC) of isopanduratin A was 4 mg/l which was much lower than that of some other natural anticariogenic agents such as sanguinarine (12 mg/l), green tea extract and carvacrol (125 mg/l), thymol (250 mg/l) and isoeugenol and eucalyptol (500 mg/l). The bactericidal test showed that isopanduratin A completely inactivated S. mutans at 20 mg/l in 1 min. Significant inhibitory activity of isopanduratin A was also observed against S. sobrinus, S. sanguinis and S. salivarius with an MIC of 4 mg/l. Damage to the cell membrane and cell wall of S. mutans by isopanduratin A was shown using transmission electron microscopy (TEM). These results suggest that isopanduratin A could be employed as a potential antibacterial agent for preventing dental caries.     

Streptococcus mutans and Streptococcus sobrinus are able to adhere and invade human gingival fibroblast cell line

Streptococcus mutans and Streptococcus sobrinus, the principal etiologic agents of caries decay of teeth, are generally acquired in oral cavity at the moment of tooth eruption. However, as S. mutans has been detected in oral cavity of predentate children, the eruption of teeth seems not to be a necessary prerequisite, suggesting that this species may be not confined to dental plaque. Here, we evaluate the ability of S. mutans and S. sobrinus in planktonic and biofilm lifestyle to adhere, invade and survive within human gingival fibroblast (HGF-1) cells. Planktonic and biofilm streptococci adhered and invaded host cells to different extents, showing higher efficiencies of biofilm than planktonic counterparts. Moreover, planktonic and biofilm streptococci showed the same percentage of survival within host cells. Transmission electron and confocal microscopy observations confirmed intracellular localization of planktonic and biofilm bacteria. The adhesion, invasion and survival abilities within human oral cells may be considered S. mutans and S. sobrinus virulence mechanisms to colonize and persist in the oral cavity in the absence of tooth surface.     

根面龋优势菌的研究

目的 研究根面龋的优势菌。方法 用选择性培养基对６０例根面龋中的细菌进行定量研究。结果 放线菌在数量上占优势。结论 支持放线菌是根面龋优势菌。     

Effects of caffeine and theophylline on the development of dental caries in rats

The purpose of this study was to evaluate the influence of caffeine and theophylline on the development of dental caries in rats. Six Wistar dams (spf), mutans streptococci free, were obtained, each with six male pups. The dams were infected by Streptococcus sobrinus 6715 and divided into three groups which received during the lactating period: (1) diet 2000; (2) diet 2000 plus caffeine (2 mg/100 g) and (3) diet 2000 plus theophylline (0.57 mg/100 g). After weaning, the pups were infected by S. sobrinus, placed in a K?nig-H?fer programmed feeder machine, and received 17 meals daily at hourly intervals, for five weeks. During this time the pups were fed with the same diet that their dams were. The percentage of S. sobrinus relative to total flora was significantly higher in the theophylline group. The results for slight (Ds) and moderate (Dm) dentine lesions, for smooth-surface and sulcal scores were statistically higher for the theophylline group than the other groups. Salivary assays did not demonstrate significant inorganic alterations in salivary composition. Caffeine and theophylline groups showed the highest ulcer score. It is concluded that caffeine does not affect the cariogenic potential of the diet, however theophylline can increase the development of dental caries, and this effect may be related to organic alterations of salivary composition.     

Antimicrobial activity of Polygonum tinctorium Lour: extract against oral pathogenic bacteria

Extract of the leaves of the indigo plant (Polygonum tinctorium Lour.) was examined for antimicrobial activity against oral pathogenic bacteria. The indigo plant extract at concentrations of 1.74–3.48 mg/ml inhibited the growth of oral cariogenic and periodontopathic bacteria. Tryptanthrin, one of the components of indigo plant extract, showed the strongest antimicrobial effect at doses ranging from 3.13–25 g/ml, indicating that this compound is one of the active ingredients. Furthermore, treatment of periodontopathic bacteria Porphyromonas gingivalis and Prevotella intermedia with the indigo plant extract for 3–8 h resulted in death of the bacteria in a dose-dependent manner, whereas Streptococcus mutans was not affected by the extract. Thus, for its antimicrobial and bactericidal effects on oral pathogens, indigo plant extract would provide useful material for preventing and treating periodontal diseases and dental caries.     

Effect of a novel type of propolis and its chemical fractions on glucosyltransferases and on growth and adherence of mutans streptococci

Flavonoids have been considered the main biologically active components in propolis. However, a new variety of flavonoid-free propolis was recently found and chemically classified as type 6. Because it showed activity against oral microorganisms, this study evaluated the effects of the crude ethanolic extract of this propolis and its chemical fractions on the activity of purified glucosyltransferases (GTFs) and on the growth and adherence of mutans streptococci. The inhibitory effect of propolis extracts on GTF activities was determined either in solution or adsorbed onto saliva-coated hydroxyapatite. Streptococcus mutans Ingbritt 1600, Streptococcus sobrinus 6715, and two clinical isolates of each species were used for antibacterial assays. Susceptibilities to the test extracts were analyzed using the agar diffusion method and by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC); the effect on bacterial adherence to a glass surface was also assessed. The activity of GTFs in solution was effectively inhibited by the ethanolic extract of propolis type 6 (EEP) (>80% inhibition at 0.5 mg/ml), hexane, and chloroform fractions (60-90% inhibition at 100 microg/ml); their inhibitory effects on surface enzymes were less pronounced. The EEP, hexane, and chloroform fractions also showed significant antibacterial activity. The data showed that propolis type 6 remarkably reduced GTF activity and inhibited mutans streptococci growth and adherence; these biological activities are associated with its nonpolar components.     

Discovery of Potent Inhibitors of Streptococcus mutans Biofilm with Antivirulence Activity

Dental caries is a bacterial infectious disease characterized by demineralization of the tooth enamel. Treatment of this disease with conventional antibiotics is largely ineffective as the cariogenic bacteria form tenacious biofilms that are resistant to such treatments. The main etiological agent for dental caries is the bacterium Streptococcus mutans. S. mutans readily forms biofilms on the tooth surface and rapidly produces lactic acid from dietary sucrose. Glucosyl transferases (Gtfs) secreted by S. mutans are mainly responsible for the production of exopolysaccharides that are crucial for the biofilm architecture. Thus, inhibiting S. mutans’ Gtfs is an effective approach to develop selective biofilm inhibitors that do not affect the growth of oral commensals. Herein, we report a library of 90 analogs of the previously identified lead compound, G43, and exploration of its structure activity relationships (SAR). All compounds were evaluated for the inhibition of S. mutans biofilms and bacterial growth. Selected compounds from this library were further evaluated for enzyme inhibition against Gtfs using a zymogram assay and for growth inhibition against oral commensal bacterial species such as Streptococcus gordonii and Streptococcus sanguinis. This study has led to the discovery of several new biofilm inhibitors with enhanced potency and selectivity. One of the leads, III_F1, showed marked reduction in buccal, sulcal, and proximal caries scores in a rat model of dental caries.     

In vitro antibacterial activity of the peptide PsVP-10 against Streptococcus mutans and Streptococcus sobrinus with and without glycocalyx

The antibacterial activity of the peptide PsVP-10 obtained from Pseudomonas sp. R10 against Streptococcus mutans and Streptococcus sobrinus was investigated. One hundred and twenty strains of S. mutans and 120 strains of S. sobrinus with and without glycocalyx were isolated from saliva samples in trypticase-yeast-cysteine-sucrose-bacitracin (TYCSB) agar. Bacterial identification was made by polymerase chain reaction. Glycocalyx production was observed in modified TYCSB agar and confirmed with a modified version of the microplate adherence assay. The minimum inhibitory concentration (MIC) of PsVP-10 bacteriocin was determined by means of the agar dilution method, and the time of bacterial death was calculated by means of colony-forming unit counts. The MIC of the bacteriocin PsVP-10 for both bacterial species with and without glycocalyx was < 2 mg/L and the time of bacterial death was less than 240 s for all the studied bacterial strains. Thus, bacteriocin PsVP-10 could be an interesting possibility to combat these cariogenic bacterial species.     

血卟啉单甲醚光动力疗法对致龋变形链球菌及龋损表面形态的影响

摘要 目的：探讨以血卟啉单甲醚(hematoporphyrin monomethyl ether,HMME)为光敏剂的光动力疗法（photodynamic therapy,PDT）在龋病预防中的作用。方法：采用变形链球菌接种Wistar大鼠磨牙,给予致龋饲料,建立龋病模型。将大鼠分为5组,分别用10 ng/L HMME-PDT（A组）、20 ng/L HMME-PDT（B组）、40 ng/L HMME-PDT（C组）、0.2 g/L氟化钠水溶液（D组）和0.9％的生理盐水(E组)处理各组大鼠磨牙,每周1次,连续4周,记录大鼠口腔变形链球菌生长情况。处死大鼠后用扫描电镜观察各组磨牙龋损区的表面形态。结果：第一周和第二周各组菌落数差异均无统计学意义（P>0.05）,第三周B组、C组菌落数低于E组（P<0.05）。第四周A组、B组和C组菌落数均低于E组（P <0.05或P <0.01）,C组菌落数低于A组和B组（P <0.05）,扫描电镜观察发现C组与D组龋损破坏程度最轻且表面相对平滑。结论：PDT具有抑制龋病的作用,将为龋病的预防提供一种新的有效途径。     

5-(Alkylsulfonyl)salicylanilides as potential dental antiplaque agents

A series of 22 5-(alkylsulfonyl)salicylanilides was synthesized and evaluated for in vitro antibacterial and antiplaque activity against Actinomyces viscosus and Streptococcus mutans, adherent microorganisms implicated in periodontal disease and dental caries. The minimum inhibitory concentrations of 25 salicylanilides (including 5-acyl-, 5-alkyl-, and 5-(alkylsulfonyl)-4'-bromo- and -4'-(trifluoromethyl)salicylanilides) were found to correlate (r = 0.94) with estimated log D values. Several salicylanilides, such as 5-(decylsulfonyl)- and 5-(dodecylsulfonyl)-4'-(trifluoromethyl)salicylanilides (15 and 19) were found to exhibit high levels of in vitro antibacterial and antiplaque activity against A. viscosus and S. mutans.     

Design and Characterization of an Acid-Activated Antimicrobial Peptide

Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype ‘acid-activated peptides’ (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.     

Low-cariogenicity of the tetrasaccharide nystose

1. The effect of nystose on cariogenic factors of mutans streptococci was investigated. 2. Nystose was not utilized as a substrate for the growth, lactic acid production, plaque formation and cellular aggregation of serotype a-g strains of mutans streptococci. 3. Furthermore, nystose did not served as a substrate for water-insoluble glucan formation by glucosyltransferase (GTase) from strains used. 4. In the cellular adherence experiment using nystose, the amount of serotype d cells that adhered to glass surface was almost zero. 5. In the presence of sucrose, nystose inhibited GTase activity and adherence of cells. 6. Finally, significantly lower caries score was observed in rats infected with Strep. sobrinus and fed with diet containing nystose as compared with sucrose.     

The in vitro dental plaque inhibitory properties of a series of N-[1-alkyl-4(1H)-pyridinylidene]alkylamines

A series of novel N-[1-alkyl-4(1H)-pyridinylidene]alkylamine hydrohalides has been prepared and evaluated as inhibitors of dental plaque formation, in vitro. Several members of the series exhibited potency ca. 9-fold greater than that of chlorhexidine vs Streptococcus sobrinus 6715-13. The di-n-octyl analogue, 11 (pirtenidine), was found to be highly efficacious against several other oral plaque-forming microorganisms and is presently undergoing preclinical evaluation.     

Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation

Many studies have demonstrated that sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents can inhibit bacterial biofilm formation. However, the mechanisms by which antimicrobial agents at sub-MICs inhibit biofilm formation remain unclear. At present, most studies are focused on Gram-negative bacteria; however, the effects of sub-MICs of antimicrobial agents on Gram-positive bacteria may be more complex. Streptococcus mutans is a major cariogenic bacterium. In this study, the S. mutans growth curve as well as the expression of genes related to S. mutans biofilm formation were evaluated following treatment with 0.5× MIC of chlorhexidine (CHX), tea polyphenols and sodium fluoride (NaF), which are common anticaries agents. The BioFlux system was employed to generate a biofilm under a controlled flow. Morphological changes of the S. mutans biofilm were observed and analysed using field emission scanning electron microscopy and confocal laser scanning microscopy. The results indicated that these three common anticaries agents could significantly upregulate expression of the genes related to S. mutans biofilm formation, and S. mutans exhibited a dense biofilm with an extensive extracellular matrix following treatment with sub-MICs of NaF and CHX. These findings suggest that sub-MICs of anticaries agents favour S. mutans biofilm formation, which might encourage dental caries progression.     

超疏水纳米膜对变形链球菌在羟基磷灰石表面黏附的影响

目的:探讨变形链球菌对表面涂有不同超疏水纳米膜的羟基磷灰石片的黏附和早期生物膜的形成。方法:比较涂有不同超疏水纳米膜和未涂有纳米膜的羟基磷灰石片对变形链球菌黏附的数量。采用体外黏附实验,了解变形链球菌在羟基磷灰石片上黏附量受表面超疏水纳米膜影响的状况。结果:3种涂有超疏水纳米膜(碳原子数量不同)的羟基磷灰石片;变形链球菌在其表面和未涂有纳米膜的羟基磷灰石片上黏附的细菌数量差异具有显著性。结论:在涂有含不同碳原子数目超疏水纳米膜的羟基磷灰石片上形成的生物膜和未涂有超疏水纳米膜的羟基磷灰石片表面形成的生物膜是不同的,且其上面黏附的细菌数量有所差别.提示超疏水纳米膜为口腔微态环境以及龋病和/或牙周病的发展具有重要意义。     

Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans

Streptococcus mutans triggers dental caries establishment by two major factors: synthesis of organic acids, which demineralize dental enamel, and synthesis of glucans, which mediate the attachment of bacteria to the tooth surface. Propolis is a natural product that may prevent dental caries. Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, is the most important botanical origin for the production of green propolis (Brazilian propolis) by honeybees. However, whether B. dracunculifolia (Bd) has an anticariogenic effect, like green propolis, remains unknown. Herein, we have made a comparative evaluation of the effects of extracts from green propolis and Bd on the glucan synthesis and acidogenic potential of S. mutans. The inhibitory effects of the extracts on bacterial acid production were evaluated through the potentiometric measurement of pH from bacterial suspensions treated with serial concentrations of both extracts. Besides presenting close inhibitory values at the same concentration range, Bd leaf rinse and green propolis extracts had similar IC(50) values (0.41 and 0.34 mg/ml, respectively). Both extracts produced a bacteriostatic effect on S. mutans cultures at a concentration of 0.40 mg/ml. Estimated inhibitory values of green propolis and Bd leaf rinse extracts on the synthesis of insoluble glucans (IC(50)=12.9 and 25.0 microg/ml, respectively) and soluble glucans (IC(50)=50.4 and 49.1 microg/ml, respectively) were not significantly different from each other at p<0.05. The results demonstrate that Bd leaf rinse and green propolis extracts have similar inhibitory effects on the S. mutans cariogenic factors evaluated herein, and allowed us to suggest that Bd leaves may be a potential source for pharmaceutical products employed for this purpose.     

电动牙刷对正畸治疗中防龋效果的影响

目的观察口腔固定正畸治疗中患者使用电动牙刷2年控制菌斑和预防龋病的效果。方法选择年龄在12～14岁的戴用固定矫治器的正畸患者60例,实验组使用电动牙刷,对照组使用保健牙刷。采用菌斑染色法记录实验前和经过2年实验后,两组患者菌斑牙面数、菌斑指数和患龋情况,并进行统计分析。结果实验组菌斑清除率为67.11%,对照组为41.58%,菌斑指数和新患龋在两组间差异有统计学意义(P<0.01)。结论使用电动牙刷比普通牙刷更能帮助固定正畸患者有效消除牙面菌斑,预防龋齿,在固定正畸中预防龋病值得推广应用。     

固定矫正青少年龋活性临床研究

目的通过检测29名12～18岁固定矫正青少年的龋活性,了解固定矫正与龋活性的关系.方法测定变形链球菌菌落数、菌斑产酸能力和唾液缓冲能力,用团体T检验的方法进行统计.结果正常青少年对照组口腔内变形链球菌菌落数明显少于固定矫正青少年组,而唾液PH值明显大于固定矫正组,统计学上有显著性差异(P＜0.01).两组菌斑产酸能力无统计学显著性差异,正常青少年唾液缓冲能力虽然高于固定矫正组,但两组之间无显著性差异(P＜0.05).结论结果显示固定矫正青少年对龋病的易感性增加.