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Controlling and reducing the formation of pathogenic biofilm on tooth surface is the key to the prevention and treatment of the biofilm-associated oral diseases. Antimicrobial peptides (AMPs), considered as possible future alternatives for conventional antibiotics, have been extensively studied for the control of bacterial infection. Due to the rapid dilution and degradation by human saliva, AMP preparations designed for oral use with longer retention and higher efficacy are in urgent need. To this end, a hydroxyapatite (HAp)-binding antimicrobial peptide (HBAMP), which is based on the fusion of a specific HAp-binding heptapeptide (HBP7) domain and a broad-spectrum antimicrobial peptide (KSLW) domain, has been developed in our laboratory. HBAMP was supposed to form a contact-active antibacterial interface on tooth surface to inhibit the formation of biofilms. In this study, we investigated its binding behaviour, antibacterial activity against bacteria in both planktonic and sessile states, enzymatic stability in human saliva, and cytocompatibility to human gingival fibroblasts (HGFs). Our findings suggest that HBAMP could adsorb on tooth surface to provide effective antibacterial activity with improved retention. This study provides a proof-of-concept on using conjugated molecules to promote antibacterial efficacy by synergistically actions of HBAMP free in solution and bound on tooth surface.

Introduction: Enterococcus faecalis is the most frequently detected species in root canal-treated teeth, and it is able to survive under starvation conditions. However, persistent periapical disease is often caused by multispecies. The aim of this study was to explore the survival of E. faecalis in starvation conditions and biofilm formation with the 4 common pathogenic species., Methods: A dual-species model of Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus in combination with E. faecalis was established and allowed to grow in phosphate-buffered saline for the examination of starvation survival. Cefuroxime sodium and vancomycin at a concentration of 100 mg/L were added into brain-heart infusion plate agar to count the 2 bacteria separately in the dual species. Scanning electron microscopy was used to observe the dual species and multiple species on the root canal dentin of bovine teeth for 48 hours. A confocal laser scanning microscope was used to show the 4 groups of dual-species biofilms on substrates with glass bottoms for 48 hours., Results: E. faecalis was more resistant to starvation in coexistence with C. albicans, S. gordonii, A. viscosus, or L. acidophilus, and S. gordonii was completely inhibited in coexistence with E. faecalis. The dual-species biofilm showed that E. faecalis formed thicker and denser biofilms on the root canal dentin and glass slides in coexistence with S. gordonii and A. viscosus than C. albicans and L. acidophilus., Conclusions: The multispecies community is conducive to the resistance to starvation of E. faecalis and biofilm formation in root canals., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed., (© 2013 Elsevier Inc. All rights reserved.)

The aim of this study was to determine the probiotic potential of autochthonous oral lactobacilli. For this, 66 strains were screened for antibacterial activity against two cariogenic strains (Streptococcus mutans and Actinomyces viscosus) and two periodontopathogenic strains (Fusobacterium nucleatum and Porphyromonas gingivalis). The inhibitory activity was investigated with the agar overlay technique. Positive results led us to explore some mechanisms of action. The ability to produce H(2)O(2) and the glycerol dehydratase gene were searched among all the strains. The gassericin A gene was checked among the Lactobacillus gasseri. All the tested strains inhibited S. mutans and A. viscosus; only one did not inhibited F. nucleatum and 52 strains inhibited slightly the growth of P. gingivalis. No inactivation of antibacterial activity was observed after treatment with proteinase K. The gene of the gassericin A was not found in any strain. Only one strain showed a 275-bp amplicon corresponding to the Glycerol Dehydratase gene. This strain has been identified by DNA 16S sequencing as a L. gasseri. Among the 66 tested strains, 7 produced hydrogen peroxide. Our findings suggest that in addition to the previous results, some of the autochthonous oral lactobacilli tested could be considered as suitable probiotics., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study investigated the antibacterial effect of a resin composite matrix with or without incorporated triclosan (0.3 wt%) on Streptococcus mutans, Actinomyces viscosus and Lactobacillus casei. In the quantitative assay, bacterial suspensions were filled into 20-μl cavities within temporary restorative resins. After 0, 4, 8, 12, 24 and 48 h of incubation, the suspensions were removed from the restoratives and the numbers of viable bacteria were determined. Bacterial suspensions incubated without restoratives served as the controls. Ten replicates were carried out for each experiment. The resin composite containing triclosan demonstrated variable degrees of antibacterial activity against the microorganisms, revealing a significant inhibitory effect on S. mutans within 12 h compared to the control. The viable counts of A. viscosus significantly decreased after 24 h. A significant reduction of L. casei was observed after 48 h. The unloaded resin composite did not reveal a marked antibacterial effect. The resin composite loaded with triclosan might be beneficial in preventing cavity contamination and minimizing the risk of pulpal irritation in the short-term.

Objectives: Antibacterial properties of restorative dental materials such as amalgam and composite resins may improve the restorative treatment outcome. This study evaluates the antibacterial properties of three composite resins: Z250, Tetric Ceram, P60 and a dental amalgam in vitro., Methods: Streptococcus mutans and Actinomyces viscosus served as test microorganisms. Three quantitative microtiter spectrophotometric assays were used to evaluate the effect of the restorative materials on: (i) early-stage biofilm using a direct contact test (DCT); (ii) planktonic bacterial growth; (iii) bacterial growth in the materials' elute. For comparison purposes, agar diffusion test (ADT) was also performed., Results: The effect of the composite resins on bacterial growth was minimal and limited to a few days only. One-week-aged composites promoted growth of S. mutans and A. viscosus. The antibacterial properties in direct contact were more potent than in planktonic bacterial growth. Amalgam showed complete inhibition of both bacteria in all phases, and the effect lasted for at least 1 week. The materials' elute had no effect on both bacterial growth with the exception of complete inhibition of S. mutans in amalgam. The later results correlated with the ADT., Conclusions: The present findings demonstrate potent and lasting antibacterial properties of amalgam, which are lacking in composite resins. This may explain the clinical observation of biofilm accumulated more on composites compared to amalgams. It follows that the assessment of antibacterial properties of poorly-soluble materials has to employ more than one assay.

Zinc-based glass polyalkenoate cements have been synthesised and their potential use in orthopaedic applications investigated. Zinc ions were released from the materials in a rapid burst over the first 24 h after synthesis, with the release rate falling below detectable levels after 7 days. Cement-implanted bone samples were prepared and the released zinc was shown, using energy dispersive X-ray analysis, to penetrate from the cement into the adjacent bone by up to 40 microm. Finally, the cements exhibited antibacterial activity against Streptococcus mutans and Actinomyces viscosus that reflected the pattern of zinc release, with the inhibition of growth greatest shortly after cement synthesis and little or no inhibition measureable after 30 days.

Background: Immunosuppressants play an essential role in transplantation therapy. In view of the side effects, e.g. gingival overgrowth, the present in vitro study was performed in order to investigate the effect of selected immunosuppressants on metabolic activities of gingival fibroblasts. Furthermore, the effect on the growth of six oral microorganisms was investigated., Methods: Human gingival fibroblasts were incubated in the presence of azathioprine (Aza), cyclosporin A (CsA), tacrolimus (Tac) or mycophenolatmofetil (Myc). PGE subset 2 release was determined by means of a specific competitive enzyme immunoassay, using monoclonal antibodies specific for PGE subset 2 (clone E2R1). The protein content was measured spectrophotometrically. A redox indicator system was employed to assess the proliferation activity. In an additional trial the growth of six strains of oral bacteria (A. viscosus T14V, S. oralis H1, S. mutans 10449, C. gingivalis DR2001, A. actinomycetemcomitans Y4, and M. micros 33270) in the presence of the immunosuppressants was measured., Results: In comparison with the controls, the PGE subset 2 release was increased by 39.3% following incubation with Aza, and by 77.0% with CsA. The protein concentrations (1 g immunosuppressant / ml medium) were reduced by 26.0% for Aza and 17.0% for Myc. Furthermore, a drug-dependent inhibition in the cell proliferation rate was noted after an incubation period of 6 hours (Aza 70.7%, CsA 78.2%, Myc 69.8%, Tac 64.0%). The most pronounced growth-inhibiting effects were observed for CsA at values ranging from 21.0% (S. mutans 10449) to 48.6% (A. viscosus T14V) growth inhibition., Conclusions: The present study with common immunsuppresants demonstrated both a medication- and dose-dependent alteration in the metabolic activity of gingival fibroblasts. Furthermore, growth-inhibitory effects on the selected bacterial strains could be observed.

Objective: To examine the effects of para-aminobenzoic acid (PABA) on the growth of Actinomyces viscosus., Methods: Different concentrations of PABA (10(-10)-10(-3) g/L) were each transferred to modified Carlsson medium. Actinomyces viscosus ATCC19246 grew in them. And the cultures were incubated at 37 degrees C anaerobically in the atmosphere of 80%N2, 10%H2, 10%CO2 for 48 h. Actinomyces viscosus OD values (lambda = 540 nm) were obtained with UV-1601. Colony forming unit (CFU) was established by growth of Actinomyces viscosus in culture when different concentrations of PABA (10(-10)-10(-3) g/L) were present., Results: Different concentrations of PABA (10(-10)-10(-4) g/L) had different stimulating effects on the growth of Actinomyces viscosus (P < 0.05). But this kind of stimulating effect declined when PABA concentration was 10(-5) g/L, and as PABA (10(-3) g/L) was present, this kind of effect was missing., Conclusion: The phenomena indicated that PABA has stimulating effect on the growth of Actinomyces viscosus, particularly when PABA is at the concentration of 10(-6) g/L.

Particulate Bioglass is a bioactive material used in the repair of periodontal defects. This material undergoes a series of surface reactions in an aqueous environment which lead to osseointegration. The aim of this study was to determine whether these reactions exerted an antibacterial effect on a range of oral bacteria. Streptococcus sanguis, Streptococcus mutans and Actinomyces viscosus were suspended in nutrient broth (NB), artificial saliva (AS) or Dulbecco's modified eagle medium plus 10% foetal calf serum (DMEM + 10%FCS), with or without particulate Bioglass. All bacteria showed reduced viability following exposure to Bioglass in all the media after 1 h. This antibacterial effect increased after 3 h. Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Actinobacillus actinomycetemcomitans were suspended in either BM broth or 40% horse serum (HS) in RPMI. A considerable reduction in viability was observed with all bacteria tested, in both media, compared to inert glass controls. In further experiments it was found that the viability of S. sanguis was significantly reduced following exposure to NB pre-incubated with Bioglass. Additionally, it was found that neutralisation of this highly alkaline solution eliminated the antibacterial effect. Moreover, a solution of NB and NaOH (of equivalent pH) exerted an antibacterial effect of similar magnitude to that of the solution pre-incubated with Bioglass. Thus, particulate Bioglass exerts an antibacterial effect on certain oral bacteria, possibly by virtue of the alkaline nature of its surface reactions. This may reduce bacterial colonisation of its surface in vivo.

This study examined the antibacterial activities of the bonding systems Syntac, EBS and Scotchbond 1, the polyacid-modified composite resins Hytac and Compoglass, and the composite resins Tetric, Z100 and Scalp-it. They were evaluated using the cariogenic bacteria Streptococcus mutans, Lactobacillus salivarius, Streptococcus sorbinus and Actinomyces viscosus in vitro with a modified cylinder drop plate agar diffusion assay. All adhesives of the dentin bonding systems and the polyacid-modified composite resins exhibited various degrees of antibacterial activity against all of the test bacteria. On the contrary, composite resins did not affect bacterial growth. The data suggest that the use of these adhesives and polyacid-modified composite resins may reduce the consequences of microleakage owing to their antibacterial properties.

Background: Subgingival plaque bacteria live within a biofilm covered with glycocalyx, and little is known of the bacterial species associated with biofilm formation at the bottom of human periodontal pockets, the so-called "plaque-free zone"(PFZ)., Methods: Seventy-seven extracted teeth from 56 patients with severe advanced adult periodontitis were examined. Porphyromonas gingivalis, Campylobacter rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella nigrescens, and Actinomyces viscosus were examined by scanning immunoelectron microscopic techniques, using both secondary and back-scattered imaging, with rabbit antibodies specific for each bacteria., Results: Secondary electron images showed that rods, filaments, and spirochete-shaped bacteria formed small aggregates in the PFZ. Some of the bacteria were covered with an amorphous film-like structure. By back-scattered electron imaging, positive reactions with anti-P. gingivalis were found in 8 of 13 samples examined, and film-like structures coated several cells of 6 positive samples examined. Labeled cells with anti-C. rectus, anti-T. denticola and anti-P. nigrescens were detected in 3 of 11, 5 of 10, and 1 of 8 samples examined. A. viscosus were found in 6 of 11 of the samples. A. viscosus tended to overlay the amorphous capsula and aggregate. F. nucleatum and A. actinomycetemcomitans were not detected in any samples examined., Conclusions: These findings indicated that P. gingivalis, C. rectus, T. denticola, P. nigrescens, and A. viscosus were present in the PFZ, and that some specified bacteria were possibly related to plaque-biofilm formation of subgingival plaque.

Purpose: To compare and contrast antibacterial activities of a baking soda-containing dentifrice, Arm and Hammer Dental Care (AHDC) with two fluoride dentifrices without baking soda (Crest and Colgate)., Materials and Methods: A biphasic approach was taken, utilizing newly-developed laboratory model systems to: (1) assess the activity of brief exposure to dentifrices on single and mixed cultures; and (2) determine the effect of multiple, short-term exposure of sucrose-colonized Streptococcus mutans to simulate cumulative activity against cariogenic plaque., Results: The short-term exposure assays revealed that S. mutans was significantly more susceptible to AHDC than either Crest of Colgate (P<0.05). Moreover, exposure of mixed suspensions of bacteria by AHDC resulted in complete killing of Actinomyces viscosus and significantly greater decreases in S. mutans (P<0.05). This enhanced bactericidal effect was not due to an alkaline pH as pH-adjusted AHDC solutions exhibited similar activity. The comprehensive in vitro plaque studies showed that exposure of colonized S. mutans to AHDC resulted in significantly greater decreases in numbers of viable cells than Crest (P<0.05). Under the conditions employed, the baking soda-containing AHDC exhibited greater antibacterial efficacy overall than the standard Crest or Colgate pastes. These studies suggest that the use of AHDC may provide additional clinical benefit as a result of the enhanced bactericidal activity.

We developed an experimental in vitro model of dental plaque to assess the potential efficacy of antiplaque agents. The model used a chemostat, which provided a continuous source of 5 species of oral bacteria grown in an artificial "saliva-like" medium. This mixture was pumped through six flow cells, each containing two types of surfaces on which plaque formed and was subsequently measured. Formation of bacterial plaque on hydroxyapatite surfaces was assessed by measurement of the DNA and protein content of the plaque film. The amount of bacterial plaque formed on germanium surfaces was measured by attenuated total reflectance (ATR/FT-IR) spectroscopy. Plaque viability was also assessed by a fluorescent staining technique. The quantity of plaque formed on both types of surfaces gradually increased with the duration of flow (from 24 to 72 h) through the cells during a 72-hour experimental period. The flow cells were then pulsed with experimental treatment solutions for 30 s, twice daily. Parallel to results of human clinical studies, the model was capable of discriminating among water, a placebo mouthrinse, and an active antimicrobial mouthrinse formulation containing 0.03% triclosan. It therefore offers a valuable alternative to animal model testing and allows for more rapid evaluations under well-controlled experimental conditions.

The purpose of this investigation was to determine whether pyrophosphate, the anticalculus component of tartar-control dentifrices, exerts antimicrobial activity against oral bacteria commonly found in supragingival plaque. Minimal inhibitory concentrations of pyrophosphate were determined for Streptococcus sanguis, Streptococcus mutans (serotype c), Actinomyces viscosus and Actinomyces naeslundii. All of the bacteria tested were susceptible to pyrophosphate with identical minimal inhibitory concentrations of 0.67% wt/vol (25 mM). Bactericidal kinetics assays revealed that both S. mutans and A. viscosus were killed by pyrophosphate, with the latter being considerably more susceptible. The mechanism of killing was not due to high ionic strength, as comparable controls showed no loss in numbers of viable cells. Brief exposure (two 5-min incubations) of S. mutans to pyrophosphate and sodium dodecyl sulfate caused pronounced inhibition of growth over the 24-h test period. Under the constraints of the conditions used, these studies indicate that pyrophosphate and sodium dodecyl sulfate can substantially inhibit the growth of oral bacteria. These compounds may affect the oral microflora of patients who routinely use tartar-control dentifrices and mouthrinses.

Antigenic relatedness among human strains of oral Actinomyces and similar isolates from cattle has been analyzed by agglutination and immunoblotting. Whole cell agglutination placed A. viscosus serotype II, A. naeslundii serotypes II and III, Actinomyces NV, and strains from numerical taxomonic clusters C1, C2, C3, C4, and C6 into a single group. A. viscosus serotype I cross-reacted weakly with this group. A naeslundii serotype I strains and the cattle isolates Actinomyces denticolens and Actinomyces howellii were distinct. The agglutination results for A. slackii were equivocal. Immunoblots of cell wall extracts developed with non-absorbed sera showed cross-reactivity (23% to 90% antigenic similarity) among all of the strains tested, including A. israelii. The range of antigenic similarities among the group which included strains of A. viscosus serotype II, the A. naeslundii serotypes, and clusters C1, C2, C3, C4, and C6 was from 39% to 89%. Immunoblotting showed that A. howellii and A. denticolens were between 39% and 72% similar to A. naeslundii and A. viscosus. Absorption of antisera with A. israelii cell walls removed antibodies recognizing antigens common to Actinomyces and made the sera more specific. Immunoblotting with absorbed sera supported the grouping and separation of strains shown by agglutination. In some cases, serotypes could be included into a specific taxonomic cluster. A. naeslundii serotype II and Actinomyces NV most closely resembled cluster C1 strains, and A. naeslundii serotype III resembled cluster C1 strains, and A. naeslundii serotype I and A. viscosus serotype I were included into clusters C5 and C7, respectively. The results support a recent proposal that strains of A. viscosus serotype II, A. naeslundii serotypes II and III, and Actinomyces NV be included into A. naeslundii genospecies 2, that A. naeslundii serotype I should be designated A. naeslundii genospecies 1, and that A. viscosus serotype I should be retained distinct from A. naeslundii, as A. viscosus.

Coaggregations between bacterial species have been widely studied in vitro but not in the mouth. A new in vivo assay was used to measure the rate and composition of indigenous plaque formation onto bovine enamel chips covered with a continuous layer of bacteria. Chips were covered with Streptococcus oralis ATCC 10557, which coaggregated with many oral species, or Streptococcus gordonii S7, which did not coaggregate with these oral species, and placed in the mouth for 4 and 24 h. There were no differences in the number of most indigenous bacterial species isolated from the two streptococcal surfaces. However, the number of Actinomyces viscosus as a proportion of total Actinomyces spp. was significantly different on the two surfaces at 24 h. With the exception of Actinomyces naeslundii and A. viscosus removed from the S7 surface, all indigenous species increased significantly in number from 4 to 24 h, irrespective of the streptococcal surface. This study demonstrated that interbacterial coaggregation had only a limited effect on in vivo plaque development. Thus suggesting that environmental factors, growth or other adherence phenomena are dominant in in vivo plaque formation.

The establishment of S. mutans together with A. viscosus was investigated in dental plaque of specific pathogen-free (SPF) rats fed different carbohydrate diets. Two Tanzanian S.mutans strains MM3 and MM24 and one Tanzanian A. viscosus strain MM13 were used for this purpose. The basic diet consisting of 32% skim-milk, 7% yeast extract and 1% soy bean oil was supplemented with either 10% lactose and 50% corn flour, or 10% glucose and 50% corn flour, or 60% amylum or 60% wheat flour. S. mutans and A. viscosus were enumerated twenty days after inoculation. S. mutans counts were high irrespective of the dietary regime. A. viscosus counts in the glucose, lactose and amylum groups were of the same magnitude and significantly higher than those of the wheat flour group. The hypothesis that the establishment of S. mutans in sucrose free diets could be facilitated by the extracellular polysaccharides produced by A. viscosus was not supported by the present data. The finding that S. mutans can establish in high number in dental plaque of SPF rats in the absence of sucrose corroborates previous reports indicating high S. mutans counts in African populations with a low sucrose intake.