Human salivary function in relation to the prevalence of Tannerella forsythensis and other periodontal pathogens in early supragingival biofilm

OBJECTIVE: Previously, we screened 149 subjects and established four groups high or low for salivary killing of oral bacteria, and for aggregation and live and dead adherence of oral bacteria (as a combined factor). Caries scores were significantly lower in both High Aggregation-Adherence groups. Subsequently, we found that supragingival total biofilm DNA, total streptococci and two major streptococcal rRNA variants also were significantly lower in the High Aggregation-Adherence groups. In this study, we looked at the effects of those differences in salivary function on three periodontal pathogens. DESIGN: Quantitative PCR was used to determine levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis (formerly Bacteroides forsythus) in stored DNA extracts of overnight supragingival biofilm collected from buccal upper central incisors (UC), lingual lower central incisors (LC) and buccal upper and lower first molars (BM) and lingual upper and lower first molars (LM) of subjects in the four groups. RESULTS: A. actinomycetemcomitans and P. gingivalis were almost completely absent from these samples. T. forsythensis was found in 11 of 35 persons at the buccal molar site. Only two of those subjects were in the High Aggregation-Adherence groups, and that difference was statistically significant. The mean quantity of T. forsythensis also was significantly lower in the High Aggregation-Adherence groups. CONCLUSIONS: The difference between the Low and High Aggregation-Adherence groups might reflect direct interactions of salivary proteins with T. forsythensis. Alternatively, the higher levels of total biofilm and total streptococci seen in the Low Aggregation-Adherence groups might create a favourable environment for early secondary colonization of T. forsythensis.     

Effects of Nidus Vespae extract and chemical fractions on glucosyltransferases, adherence and biofilm formation of Streptococcus mutans

Nidus Vespae (the honeycomb of Polistes olivaceous, P. japonicus Saussure and Parapolybiavaria fabricius) have been extensively used in traditional Chinese medicine, given their multiple pharmacological activities, including antimicrobial, anti-inflammatory, anti-virus, anti-tumor and anesthetic properties. The present study evaluated the anti-glucosyltransferases (GTFs) activity, anti-adherence and anti-biofilm properties of 95% ethanol/water extract, cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol fractions of Nidus Vespae. Chloroform/methanol fraction showed a remarkable capacity for inhibiting the adherence of Streptococcus mutans ATCC 25175 to saliva-coated hydroxyapatite disc (S-HA) at sub-MC concentrations. In addition, the Nidus Vespae extract and chemical fractions significantly inhibited the activity of cell-associated and extracellular GTFs at sub-MIC concentrations, and the chloroform/methanol fraction was the most effective one. For the anti-biofilm activity assays, minimum biofilm inhibition concentrations (MBIC50) and minimum biofilm reduction concentrations (MBRC50) were determined using the microdilution method. The chloroform/methanol fraction showed the highest anti-biofilm activities with a MBIC50 of 8mg/ml and a MBRC(50) of 16mg/ml against Streptococcus mutans ATCC 25175. The significant inhibition of GTFs activity and biofilm formation demonstrated by Nidus Vespae shows it to be a promising natural product for the prevention of dental caries.     

Prevalence and distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitans in chronic periodontitis Brazilian subjects

Objective

Previous studies have suggested that Aggregatibacter actinomycetemcomitans is involved in the aetiology of aggressive periodontitis as well as chronic periodontitis. In addition, some authors have also reported that serotype-specific antigens of A. actinomycetemcomitans determine the severity of disease. This study aimed to elucidate the prevalence of A. actinomycetemcomitans and the distribution of A. actinomycetemcomitans serotypes in Brazilian subjects with chronic periodontitis.

Design

A total of 486 individuals were enrolled in this survey. All patients received clinical examinations that included periodontal pocket depth, clinical attachment loss, plaque, and gingival indexes. Subgingival samples were taken for microbial analysis. The genomic DNA of A. actinomycetemcomitans was provided by PCR.

Results

Out of 486 subjects examined, A. actinomycetemcomitans was isolated in 85 (17.5%) individuals. Out of 85 positive samples, 68 were infected by at least 1 serotype, 7 by mixed infection, and 10 were non-serotyped. Serotypes d and f were not detected. Serotype c showed the highest prevalence (52.9%), followed by serotype a (31.8%).

Conclusions

Intragroup analysis revealed that, in slight/moderate periodontitis, serotypes c and a were significantly more prevalent than serotypes b and d-f; the prevalence of serotype c in severe periodontitis was significantly greater than that of serotypes a and b. Our data were similar in Asian and Eurasian populations.     

In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions

An understanding of biofilm behavior of Candida species under different environmental conditions is key to the development of effective preventive measures for candidal infections. Hence in this study we assessed the impact of the environmental milieu on Candida biofilm formation using polystyrene, flat-bottomed 96-well microtiter plates. A total of 20, comprising 10 clinical isolates each of Candida albicans and, non-albicans species of Candida were compared for their biofilm forming ability both under aerobic and anaerobic conditions, and static and dynamic conditions. XTT reduction assay was used to quantify the sessile growth. Biofilm formation of all 10 C. albicans isolates differed significantly between dynamic and static states under both atmospheric conditions (P<0.05). For non-albicans Candida species, a significant difference in biofilm growth between dynamic and static states was noted only when incubated aerobically (P<0.05), and no significant difference in biofilm formation was noted between aerobic and anaerobic conditions. Scanning electron microscopy revealed that C. albicans produced a compact multilayered biofilm embedded in noticeably higher quantity of extracellular polymeric matrix in aerobic/dynamic conditions compared with anaerobic/static conditions. Our data indicate that biofilm formation of C. albicans and non-albicans Candida species is modulated by hydrodynamic conditions and ambient oxygen gradients. However, further work is required to fully elucidate how Candida biofilms persist within the oral milieu under such challenging ecological pressures.     

Effect of aqueous extract from Neem (Azadirachta indica A. Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans

OBJECTIVE: Azadirachta indica, a Meliaceae family tree, has been used in India for many years in the treatment of several diseases in medicine and dentistry. Current research analyses the effects of the leaf aqueous extract from Azadirachta indica (Neem) on the adhesion, cell surface hydrophobicity and biofilm formation, which may affect the colonisation by Candida albicans. METHODS: Azadirachta indica extract was tested in vitro on strains of Candida albicans 12A and 156B. Changes in hydrophobicity were reported in assays of yeast adhesion to hydrocarbons, in biofilm formation with glucose and in the adhesion of the microorganisms on light cured composite resin. Assays involved enumeration of candidal colony-forming units together with scintillation counting of radiolabelled Candida and compared to a solution of chlorhexidine digluconate 0.125% widely used in dentistry. RESULTS: Yeast growth in Neem extract was not inhibited in concentrations ranging from 0.1mg/ml. A statistically significant increase (p<0.05) in cell surface hydrophobicity was evident for the two strain tested and there was also an associated increase in biofilm formation after contact with Neem extract in concentration 0.01 g/ml. Decrease in adhesion capacity of cells to composite resin was also recorded. CONCLUSION: An anti-adhesive mechanism of action by Azadirachta indica is proposed based on the results observed.     

Effect of low dose Actinobacillus actinomycetemcomitans lipopolysaccharide pretreatment on cytokine production by human whole blood

BACKGROUND AND OBJECTIVE: Periodontal disease is known to influence the systemic condition in various ways, and the bacteria and their products, such as lipopolysaccharides (LPS), may spread from periodontal lesions via the systemic circulation to affect distant organs. The level of LPS in plasma from such patients is reported to be very low, and this low level of LPS is suspected to have priming or desensitizing effect. Thus, we investigated the effects of low dose LPS pretreatment on LPS-dependent cytokine production by whole blood cells ex vivo. METHODS: Blood samples obtained from seven systemically and periodontally healthy individuals were pretreated with or without 5 pg/ml Actinobacillus actinomycetemcomitans LPS, followed by further stimulation with 1 ng/ml A. actinomycetemcomitans LPS. The concentrations of interleukin-1 beta (IL-1beta), IL-6, IL-10 and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatants were then determined using enzyme-linked immunosorbent assay (ELISA). In addition, intracytoplasmic cytokine staining of whole blood cells was performed for flow cytometry. RESULTS: Pretreatment with 5 pg/ml A. actinomycetemcomitans LPS significantly enhanced the production of IL-1beta and IL-6 from whole blood when further induced by 1 ng/ml LPS (1.72 times higher for IL-1beta, 2.18 times higher for IL-6 than without pretreatment). The pretreatment did not enhance the production of either TNF-alpha or IL-10. Intracytoplasmic staining showed that the monocyte fraction was primarily involved in producing IL-1beta and IL-6. Flow cytometric analysis revealed that pretreatment increased the number of IL-1beta and IL-6 producing cells as well as mean fluorescence intensity of the stained cells. CONCLUSION: A low dose of bloodstream LPS found in periodontitis patients appears to be sufficient to prime monocytes, and may be capable of affecting the systemic responses of immune and inflammatory cells.     

Microbial biofilm formation in DUWS and their control using disinfectants

OBJECTIVES: Due to the presence of extended narrow bore tubing and long periods of stagnation, dental unit water systems (DUWs) can be prone to relatively high levels of microbial contamination, including the formation of biofilm and the presence of opportunistic pathogens, irrespective of the source and quality of the inflowing water. Whilst the European Union (EU) has yet to set a definitive microbiological guideline, the American Dental Association (ADA) has set a maximum of <200 colony forming units (cfu)/ml for DUWs water in the USA. The objective of this review is to discuss why microbial contamination and biofilms are so prevalent in DUWs, as well as the role of disinfectants and their potential for achieving microbial water quality levels recommended by the ADA. STUDY SELECTION: The review outlines the principal factors responsible for biofilm formation in DUWs and a number of mechanisms used for microbial control. SOURCES: The source material contained in this review is taken from the peer-reviewed literature. DATA: A variety of disinfectants are available for use, but controlled laboratory and clinical studies have shown that they can vary markedly in their efficacy and suitability for use. Some products have been shown to successfully remove biofilm and consistently reduce the microbial load of out-flowing water to <200 cfu/ml. CONCLUSIONS: The effective delivery of approved disinfectants can control the level of microorganisms in DUWs at acceptable levels.     

Relationship of biofilm formation and gelE gene expression in Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment

Introduction

Enterococcus faecalis is known to be the most frequently detected species in root canals with failed endodontic treatment. Many studies are available on biofilm formation and the expression of virulence factors such as gelatinase (gelE) in E. faecalis. However, the relationship of biofilm formation and the expression of gelE in E. faecalis recovered from root canals undergoing orthograde retreatment is not well understood.

Methods

E. faecalis was isolated from clinical samples of root canal retreatment, and the expression of gelE in E. faecalis was assessed. Automatic microplate reader and scanning electron microscopy were used to investigate the biofilm formation ability of E. faecalis isolates. Real-time quantitative polymerase chain reaction was used for detecting the expression of gelE in biofilm-positive and biofilm-negative E. faecalis isolates.

Results

The detection rate of E. faecalis in the root canal retreatment cases was 39.26%. An automatic microplate reader showed that most isolates were able to form biofilms, and the biofilm formation ability of strains isolated from the teeth without a sinus tract was better than that with a sinus tract (P < .05). The expression of gelE was stronger in the cases of apical radiolucency than in those without the symptom (P < .05). The expression of gelE was higher in the biofilm-positive than in biofilm-negative strains (P < .05).

Conclusions

Biofilm formation in E. faecalis was facilitated in the cases without a sinus tract. In the cases of apical radiolucency and in the biofilm-positive strains, the expression of gelE was higher.     

Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro

Objectives

To evaluate the influence of desensitising paste containing 8% arginine and calcium carbonate (Ar-Ca) on biofilm formation on dentine.

Methods

Dentine discs were cut from extracted third molars and divided into the following three groups: no treatment, pumice treatment and Ar-Ca treatment. Surface topography and roughness were examined using scanning electron microscopy (SEM) and non-contact 3D surface profiler. After sterilisation, samples were incubated with Streptococcus mutans (S. mutans) for 4 h, 24 h and 72 h. Bacterial adhesion and biofilm formation were analysed using SEM, whereas MTT and lactic acid production assays were used to analyse the metabolic activity of S. mutans.

Results

After polishing with either pumice or Ar-Ca, the surfaces of the samples became smoother than in the control group. The Ra values of the three experimental groups decreased significantly to 0.43 μm, 0.3 μm and 0.26 μm, respectively. Compared to the control group, fewer bacteria adhered to the dentine surface in the Ar-Ca group, while biofilm thickness decreased significantly for both groups after incubating for 24 h and 72 h. MTT and lactic acid production levers also showed a significant reduction in the Ar-Ca group.

Conclusions

Ar-Ca appears to present antibiofilm efficacy and may provide a promising approach to combat bacterial infection in hypersensitive dentinal lesions.

Clinical significance

As a clinical application of desensitising polishing paste, the paste containing 8% arginine and calcium carbonate could also inhibit the biofilm formation effectively.     

伴放线放线杆菌外膜蛋白的研究进展

伴放线放线杆菌与牙周炎,特别是与局限性侵袭性牙周炎有着密切的关系.伴放线放线杆菌外膜蛋白作为其重要毒力因子,在牙周病的发病中起着重要的作用.本文就近年来有关伴放线放线杆菌外膜蛋白的结构特征、外膜蛋白的表现型、外膜蛋白与血清型、外膜蛋白的致病性等研究进展作一综述.     

Inhibition of Streptococcus mutans adherence and biofilm formation using analogues of the SspB peptide

Objective

Streptococcus gordonii is a pioneer colonizer of the enamel salivary pellicle that forms biofilm on the tooth surfaces. Recent reports show the surface protein analogue peptide {400 (T) of SspB 390-402 is substituted to K forming SspB (390-T400K-402)} from S. gordonii interacts strongly with salivary receptors to cariogenic bacteria, Streptococcus mutans. To characterize the analogue peptide biological activities, we investigated its binding and inhibiting effects, and the role of its amino acid moities.

Methods

We measured binding activity of analogue peptides to salivary components using the BIAcore assay; assayed inhibition activities of peptides for bacterial binding and growth on saliva-coated hydroxyapatite beads (s-HA); and describe the peptides interfering with biofilm formation of S. mutans on polystyrene surfaces.

Results

The SspB (390-T400K-402 and -401) peptides significantly bound with salivary components and inhibited the binding of S. mutans and S. gordonii to s-HA without bactericidal activity; but did not inhibit binding of Streptococcus mitis, a beneficial commensal. Further, the lack of D and E-L at position 390 and 401-402 in the peptide, and substituted peptide SspB (D390H- or D390K-T400K-402) did not bind to salivary components or inhibit binding of S. mutans. The SspB (390-T400K-402) peptide inhibited biofilm formation on salivary components-coated polystyrene surfaces in absence of conditioned planktonic cells.

Conclusions

We found constructing the peptide to include positions 390(D), 400(K) and 401(E), two surface positive and negative connective charges, and at least 12 amino acids are required to bind salivary components and inhibit the binding of S. mutans and S. gordonii.     

Environmental cues and gene expression in Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans

Microorganisms typically adapt to environmental cues by turning on and off the expression of virulence genes which, in turn, allows for optimal growth and survival within different environmental niches. This adaptation strategy includes sensing and responding to changes in nutrients, pH, temperature, oxygen tension, redox potential, microbial flora, and osmolarity. For a bacterium to adhere to, penetrate, replicate in, and colonize host cells, it is critical that virulence genes are expressed during certain periods of the infection process. Thus, throughout the different stages of an infection, different sets of virulence factors are turned on and off in response to different environmental signals, allowing the bacterium to effectively adapt to its varying niche. In this review, we focus on the regulation of virulence gene expression in two pathogens which have been implicated as major etiological agents in adult and juvenile periodontal diseases: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. Understanding the mechanisms of virulence gene expression in response to the local environment of the host will provide crucial information in the development of effective treatments targeted at eradication of these periodontal disease pathogens.     

Influence of different biomaterials on the viability of Aggregatibacter actinomycetemcomitans

Objectives

The aim of the present in vitro study was to evaluate the effects of different biomaterials used for regenerative periodontal surgery on the growth of the periodontopathogen Aggregatibacter actinomycetemcomitans.

Methods

Three commercially available biomaterials of synthetic origin (hydroxyapatite/beta-tricalcium phosphate, nanostructured hydroxyapatite paste, oily calcium hydroxide suspension), a bovine-derived xenograft as well as an enamel matrix derivative (EMD) were added in different concentrations to calibrated suspensions of A. actinomycetemcomitans ATCC 43718/33384 (serotype b/c). Equal aliquots (0.1 ml) for the viability assay were taken after 5 min, 1 h, 3 h, 8 h and 24 h, plated on blood agar and incubated in an anaerobic environment for 48 h at 37 °C. Viable cell counts were expressed as colony forming units (cfu)/0.1 ml.

Results

The results demonstrated that none of the investigated biomaterials could inhibit the growth of A. actinomycetemcomitans serotype b. A marked growth reduction of A. actinomycetemcomitans serotype c was observed in the presence of oily calcium hydroxide suspension and nanostructured hydroxyapatite. In contrast, no significant growth inhibition could be observed in the presence of hydroxyapatite/beta-tricalcium phosphate, enamel matrix derivative and bovine-derived xenograft.

Conclusions

The results of the present study suggest that none of the investigated biomaterials possesses antimicrobial properties against A. actinomycetemcomitans serotype b. Therefore, the use of these biomaterials for regenerative procedures should be weighted critically in the presence of A. actinomycetemcomitans serotype b.     

臭氧水对伴放线放线杆菌的灭活效果观察

目的探讨臭氧水对牙周可疑致病菌伴放线放线杆菌（Aa）的灭活效果。方法采用悬液定量杀菌方法和和化学方法在实验室进行观察。用4、8、15mg／L的臭氧水分别对悬液中A。作用1、2、3min。结果当臭氧水浓度为4mg／L对悬液中An没有杀灭作用。当臭氧水浓度为8mg儿时,对悬液中An作用1min,杀灭率为57％,当浓度上升至15mg／L时,对悬液中Aa作用1min．杀灭率上升至98％．而延长杀菌时间至3min．杀菌率维持在97％～99％。结论在25℃的室温条件下．臭氧水浓度达到15mg／L．臭氧水温控制在15℃～18℃时对悬液中Aa有快速、有效的杀灭作用。     

Antiadherent activity of Schinus terebinthifolius and Croton urucurana extracts on in vitro biofilm formation of Candida albicans and Streptococcus mutans

Objective

To evaluate the antiadherent property of crude, methanol and acetate methanol extract fractions from Schinus terebinthifolius and Croton urucurana in hydroalcoholic (HA) and dimethylsulfoxide (DMSO) solvents on in vitro biofilms formed by Streptococcus mutans and Candida albicans strains.

Design

The minimal concentration of adherence (MICA) was determined to evaluate the antiadherent potential of extracts on the in vitro biofilm formation. The extracts of plants were subjected to thin layer chromatography (TLC) in order to detect what class of compounds was responsible for the antiadherent activity. Data were estimated by analysis of variance (ANOVA) complemented by Tukey test level of significance set at 5%.

Results

Both plants demonstrated inhibition of S. mutans and C. albicans on in vitro biofilm formation. The biofilms of C. albicans were more efficiently inhibited by the S. terebinthifolius fraction of acetate–methanol and methanol in hydroalcoholic solvents (p < 0.05). The S. mutans biofilms adherence was best inhibited by the S. terebinthifolius crude extract and its methanolic fraction, both in hydroalcoholic solvent (p < 0.05). TLC of crude extracts and fractions of S. terebinthifolius detected the presence of several active compounds, including phenolic compounds, anthraquinones, terpenoids, and alkaloids. C. urucurana extracts confirmed activity for both microorganisms (p < 0.05). However, higher concentrations were needed to achieve antiadherent activity, mainly to inhibit in vitro biofilm formation of C. albicans.

Conclusion

The antiadherent potential of both plants on in vitro biofilms formed by C. albicans and S. mutans were confirmed, suggesting the importance of studies about these extracts for therapeutic prevention of oral diseases associated with oral biofilms.     

伴放线放线杆菌形态变化对菌体表面疏水性影响的研究

目的研究伴放线放线杆菌形态变化对菌体表面疏水性的影响。方法采用碳氢化合物法检测伴放线放线杆菌粗糙型和光滑型的菌体表面疏水性,观察同一菌株不同表型疏水性的变化。结果伴放线放线杆菌粗糙型和光滑型菌体表面具有疏水性。14株粗糙型伴放线放线杆菌菌体表面疏水率高于4株光滑型,差异有统计学意义（P〈0．05）。4株同源的粗糙型与其光滑型转变株比较得出除1株外,其余3株菌两种表型的菌体表面疏水率差异无统计学意义（P〉0．05）。结论伴放线放线杆菌形态变化可引起菌体表面疏水性的改变,粗糙型转变为光滑型后菌体表面疏水性减弱。     

Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars

The pathogenesis of both superficial and systemic candidiasis is closely dictated by properties of the yeast biofilms. Despite extensive investigations on bacterial biofilms, the characteristics of candidal biofilms, and various factors affecting this process remain to be determined. Therefore we examined the effect of human whole saliva and dietary sugars, glucose and galactose on the adhesion and biofilm formation of Candida albicans. Biofilms of C. albicans isolate 192 887 g were developed on polystyrene, flat-bottomed 96-well microtiter plates and monitored using ATP bioluminescence and tetrazolium (XTT) reduction assays as well as the conventional colony forming unit (CFU) evaluation. Our data showed that both the ATP and the XTT assays strongly correlated with the CFU assay (ATP versus CFU: r = 0.994, P = 0.006; XTT versus CFU: r = 0.985, P = 0.015). Compared with a glucose-supplemented (100 mM) medium, galactose containing (500 mM) medium generated consistently lower levels of both candidal adhesion and biofilm formation (all P < 0.05), but a higher pace of biofilm development over time (96 h). Whist the presence of an immobilised saliva coating had little effect on either the candidal adhesion or biofilm formation, the addition of saliva to the incubation medium quantitatively affected biofilm formation especially on day 3 and 4, without any significant effect on yeast adhesion. To conclude, biofilm formation of C. albicans within the oral milieu appears to be modulated to varying extents by dietary and salivary factors and, further investigations are required to elucidate these complex interactions.     

Selective killing of Aggregatibacter actinomycetemcomitans by ciprofloxacin during development of a dual species biofilm with Streptococcus sanguinis

Objectives

Periodontal disease is associated with a pathogen-induced transition to a chronic destructive inflammatory response. Since commensals may either passively or actively contribute to immune homeostasis, therapies aimed at selectively reducing the competitive advantage of pathogens may be effective supplements to traditional methods. We developed an in vitro system to grow biofilms composed of the pathogen (Aggregatibacter actinomycetemcomitans) and the commensal (Streptococcus sanguinis). We used the biofilm model to determine the feasibility of selectively killing the pathogen using the fluoroquinolone, ciprofloxacin.

Design

Biofilms were exposed to relevant ciprofloxacin doses during the first 24 h of development, with subsequent removal of the ciprofloxacin for a 24 h period. Biofilm growth was assessed by confocal laser scanning microscopy, crystal violet staining and DNA abundance.

Results

Exposure to 0.01 mg/L or 0.5 mg/L ciprofloxacin significantly reduced the microcolony size and cell surface density of A. actinomycetemcomitans in the dual species biofilm over a 24 h period whilst allowing uninhibited S. sanguinis biofilm formation. A. actinomycetemcomitans biofilm development was insignificant over a subsequent 24 h period after removal of the ciprofloxacin indicating that A. actinomycetemcomitans cells were killed.

Conclusions

A. actinomycetemcomitans residing in a dual species biofilm with the commensal, S. sanguinis can be selectively killed, or at least rendered metabolically inactive, by treatment with ciprofloxacin. The dual species biofilm model will be a useful tool for designing in vivo studies to determine the efficacy of selective killing agents as an adjunct treatment of localized aggressive forms of periodontal disease.     

Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis. This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A. actinomycetemcomitans. It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components. This study focuses on the effect of histatins, components of the nonimmune oral defense system, on leukotoxin activity. Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A. actinomycetemcomitans strain Y4. PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic histatin 5 (0-500 microM). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals. Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM. When PMNs were preincubated with histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed. This observation suggests a mechanism of inhibition whereby histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction. The inhibitory effect of histatin 5 on leukotoxic activity may suggest a new biological function of histatins in the oral cavity as a naturally occurring secondary antibiotic.     

Hydrophilicity of dentin bonding systems influences in vitro Streptococcus mutans biofilm formation

Objective

To evaluate in vitro Streptococcus mutans (S. mutans) biofilm formation on the surface of five light-curing experimental dental bonding systems (DBS) with increasing hydrophilicity. The null hypothesis tested was that resin chemical composition and hydrophilicity does not affect S. mutans biofilm formation.

Methods

Five light-curing versions of experimental resin blends with increasing hydrophilicity were investigated (R₁, R₂, R₃, R₄ and R₅). R₁ and R₂ contained ethoxylated BisGMA/TEGDMA or BisGMA/TEGDMA, respectively, and were very hydrophobic, were representative of pit-and-fissure bonding agents. R₃ was representative of a typical two-step etch-and-rinse adhesive, while R₄ and R₅ were very hydrophilic resins analogous to self-etching adhesives. Twenty-eight disks were prepared for each resin blend. After a 24 h-incubation at 37 °C, a multilayer monospecific biofilm of S. mutans was obtained on the surface of each disk. The adherent biomass was determined using the MTT assay and evaluated morphologically with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).

Results

R₂ and R₃ surfaces showed the highest biofilm formation while R₁ and R₄ showed a similar intermediate biofilm formation. R₅ was more hydrophilic and acidic and was significantly less colonized than all the other resins. A significant quadratic relationship between biofilm formation and hydrophilicity of the resin blends was found. CLSM and SEM evaluation confirmed MTT assay results.

Conclusions

The null hypothesis was rejected since S. mutans biofilm formation was influenced by hydrophilicity, surface acidity and chemical composition of the experimental resins. Further studies using a bioreactor are needed to confirm the results and clarify the role of the single factors.