Antimicrobial efficiency of mouthrinses versus and in combination with different photodynamic therapies on periodontal pathogens in an experimental study.

BACKGROUND AND OBJECTIVE: In the therapy of destructive periodontal disease, chemical antimicrobial agents and increasingly photodynamic therapy (PDT) play an important adjunctive role to standard mechanical anti-infective treatment procedures. However, both antiseptic methods have their shortcomings in terms of eliminating periodontal pathogens. The aim of the study was to compare the antibacterial efficacy of different antiseptic mouthrinses, of a conventional and a new, modified PDTplus as well as of the different antiseptic mouthrinses combined with either the conventional or the modified PDTplus against periopathogens. MATERIAL AND METHODS: Six representative periodontitis-associated bacterial strains were grown for 24 h under anaerobic conditions. After mixing the individual cell pellets they were exposed to 10 different antiseptic mouthrinse formulations: chlorhexidine (0.2%, 0.06%, CHX); CHX + cetylpyridinium chloride (each 0.05%); sodium hypochlorite (0.05%); polyhexanide (0.04%, PHMB1; 0.1%, PHMB2); octenidine dihydrochloride (0.1%); fluoride (250 ppm); essential oils; povidone iodine (10%); and saline (0.9%, NaCl) as control. Furthermore, the bacteria were treated with conventional PDT based on light-emitting diodes and a new modified photodisinfection combining photosensitizer with hydrogen peroxide to PDTplus also based on light-emitting diodes. In addition to the single treatments, a combined application of antiseptic exposure followed by use of PDT or PDTplus was performed. The microbial viability was characterized by analyzing colony growth and fluorescence-based vitality proportions. RESULTS: Nearly all mouthrinses caused a statistically significant growth inhibition. The most effective antiseptics, CHX (0.2%), CHX/cetylpyridinium chloride and octenidine dihydrochloride, inhibited bacterial growth completely. Conventional PDT resulted in moderate reduction of colony growth. The modified PDTplus achieved maximum antimicrobial effect. The combination of antiseptic exposure and PDT against periopathogens predominantly increased antibacterial efficacy compared to the single applications. The mouthrinse containing essential oil seemed to interfere with PDT. CONCLUSION: A combination therapy of preceding chemotherapeutical exposure and subsequent photodisinfection may be a more effective and promising antibacterial treatment than single applications of the antiseptic methods. The modified PDTplus using oxygen-enriched toluidine showed a superior antibacterial effect on periodontal pathogens to conventional PDT and to the majority of the investigated mouthrinses.

Antibacterial Efficacy of a Propolis Toothpaste and Mouthrinse Against a Supragingival Multispecies Biofilm.

PURPOSE: To determine in vitro the antibacterial properties of propolis toothpaste and mouthrinse against an in vitro multispecies biofilm model. MATERIALS AND METHODS: Six-species biofilms grown anaerobically on pellicle-coated hydroxyapatite disks were fed with glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in 37°C saliva between feedings for up to 64.5 h. At each interval, biofilms were exposed to six different slurries and solutions, including: 1) toothpaste without propolis, 2) toothpaste with propolis, 3) toothpaste with chlorhexidine, 4) mouthrinse with propolis, 5) mouthrinse with chlorhexidine, 6) saline solution (control). Afterwards, biofilms were harvested and the number of colony forming units were determined (CFU). The results were analysed using ANOVA, followed by the Bonferroni test at a 5% significance level. RESULTS: The strongest CFU reduction was shown after treatment with 0.12% chlorhexidine (p<0.0004). When comparing the different toothpastes, there was no statistically significant difference (p<0.05) in CFU reduction. However, they all showed a significant reduction in CFU of more than one log-step vs the saline control group. Nevertheless, the propolis-containing mouthrinse showed no significant reduction in CFU. CONCLUSION: All toothpastes under investigation displayed some growth inhibition in this supragingival biofilm model, which accounted for an approximately 80%-88% linear reduction. However, the propolis mouthwash had no effect.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

Red wine and oenological extracts display antimicrobial effects in an oral bacteria biofilm model.

The antimicrobial effects of red wine and its inherent components on oral microbiota were studied by using a 5-species biofilm model of the supragingival plaque that includes Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar. Microbiological analysis (CFU counting and confocal laser scanning microscopy) of the biofilms after the application of red wine, dealcoholized red wine, and red wine extract solutions spiked or not with grape seed and inactive dry yeast extracts showed that the solutions spiked with seed extract were effective against F. nucleatum, S. oralis and A. oris. Also, red wine and dealcoholized wine had an antimicrobial effect against F. nucleatum and S. oralis. Additional experiments showed almost complete and early degradation of flavan-3-ol precursors [(+)-catechin and procyanidin B2] when incubating biofilms with the red wine extract. To our knowledge, this is the first study of antimicrobial properties of wine in an oral biofilm model.

No evidence of triclosan-resistant bacteria following long-term use of triclosan-containing toothpaste.

BACKGROUND AND OBJECTIVE: There is a paucity of data in relation to the possible emergence of triclosan (TCS)-resistant bacteria following long-term exposure to TCS toothpaste. Therefore, this study investigated whether long-term continuous exposure to TCS in toothpaste selects for TCS-resistant bacteria within the oral biofilm. MATERIAL AND METHODS: Dental plaque samples were collected from 40 individuals during year 5 of a randomised controlled trial. Participants had been randomly assigned to use TCS (3000 µg/mL TCS) (n = 18) or placebo toothpaste (n = 22). Diluted plaque samples were plated on to Wilkins-Chalgren agar plates containing 5% (v/v) laked sheep red blood cells and TCS (concentrations ranging from 25 to 150 µg/mL) and incubated at 37 °C under microaerophilic and anaerobic conditions for 2-10 d. Selected bacterial isolates were identified by partial 16S rDNA sequencing and TCS minimum inhibitory concentration (MIC) determined for each isolate. RESULTS: At 3000 µg/mL TCS no growth was observed under microaerophilic or anaerobic conditions in either group. The MICs of TCS for all isolates ranged from 125 to 1000 µg/mL in both groups. Species common to both groups had similar MICs. Veillonella parvula and Campylobacter gracilis were the most frequent isolates from both groups, with similar MICs in both groups. CONCLUSION: The use of TCS-containing toothpaste did not appear to lead to an increase in MIC of TCS of oral bacterial isolates.

Antibacterial effects of blackberry extract target periodontopathogens.

BACKGROUND AND OBJECTIVE: Antimicrobial agents provide valuable adjunctive therapy for the prevention and the control of oral diseases. Limitations in their prolonged use have stimulated the search for new, naturally occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the antibacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens. MATERIAL AND METHODS: The effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated using the colorimetric water-soluble tetrazolium-1 assay. The bactericidal effects of whole BBE against Fusobacterium nucleatum were determined by quantitating the numbers of colony-forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells. RESULTS: BBE at 350-1400 µg/mL reduced the metabolic activity of Porphyromonas gingivalis, F. nucleatum and Streptococcus mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in the numbers of CFUs following exposure to BBE for as little as 1 h, indicative of its bactericidal properties. An anthocyanin-enriched fraction of BBE reduced the metabolic activity of F. nucleatum, but not of P. gingivalis or S. mutans, suggesting the contribution of species-specific agents in the whole BBE. Oral epithelial cell viability was not reduced following exposure to whole BBE (2.24-1400 µg/mL) for ≤ 6 h. CONCLUSION: BBE alters the metabolic activity of oral periodontopathogens while demonstrating a minimal effect on commensals. The specific antibacterial properties of BBE shown in this study, along with its previously demonstrated anti-inflammatory and antiviral properties, make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.

Effect of an essential oil-containing antimicrobial mouthrinse on specific plaque bacteria in vivo.

AIM: This study was conducted to investigate the effect of rinsing with an essential oil-containing mouthrinse on levels of specific supra and subgingival bacteria in subjects with gingivitis. MATERIAL AND METHODS: Fifteen subjects meeting entry criteria completed this randomized, controlled, double-blind, crossover study. Subjects were required to have >or=1000 target organisms per millilitre in pooled samples from two subgingival sites. Following sampling of supra and subgingival plaque, subjects began twice-daily rinsing for 14 days with either an essential oil-containing mouthrinse (Cool Mint Listerine Antiseptic) or a negative control. Supra and subgingival plaque was again sampled on day 15, and the procedure repeated after a 1-week washout period with subjects using the alternate rinse. RESULTS: Compared with the negative control, the essential oil mouthrinse produced significant reductions in supragingival plaque levels of Veillonella sp., Capnocytophaga sp., Fusobacterium nucleatum, and total anaerobes ranging from 52.3 to 88.5% (p<0.001 except for Veillonella, p=0.002); respective reductions in subgingival plaque ranged from 54.1 to 69.1% (p<0.001). CONCLUSIONS: Rinsing with the essential oil mouthrinse can have an impact on the subgingival plaque flora. This study provides additional evidence indicating that reduction in supragingival plaque can reduce levels of subgingival plaque.

Efficacy of gasiform ozone and photodynamic therapy on a multispecies oral biofilm in vitro.

Ozone gas and photodynamic therapy (PDT) have been claimed to be antimicrobially effective. This study assessed their antimicrobial potential in vitro. Mature six-species oral biofilms were treated as follows (n = 9 per group): (i) a 60-s application of gasiform vacuum-ozone or vacuum alone (on wet or air-dried biofilm samples); (ii) PDT (i.e. methylene blue in combination with or without a diode soft laser, and a soft laser alone); or (iii) antimicrobial solutions: immersion of biofilms for 60 s in 0.2 and 2% chlorhexidine or in 0.5 and 5% hypochlorite solution. Treatment with chlorhexidine or hypochlorite served as a positive control, whereas untreated samples served as negative controls. Colony-forming units on blood agar were counted. Only the 5% hypochlorite solution was able to totally eliminate the microorganisms in the biofilm. The observed reduction of viable counts by vacuum-ozone application and PDT was less than one log(10) step. Under the conditions of the current study, gasiform ozone and PDT had a minimal effect on the viability of microorganisms organized in a cariogenic biofilm.

In vivo evaluation of the effect of essential oil-containing oral strips on salivary bacteria using the checkerboard method.

OBJECTIVE: The aim of this study was to evaluate the antimicrobial effect of essential oil-containing oral strips on different species of the oral microbiota. METHODOLOGY: Saliva samples were collected from 20 subjects with good oral health, diluted and plated onto blood agar medium. The subjects were asked to place the strip (Listerine PocketPaks) on the tongue allowing it to dissolve. After 30 minutes, new saliva samples were collected again and the plates with the samples were incubated under anaerobic conditions at 37 degrees C for seven days. Colony counts (CFU/mL) were determined for each sample. The colonies on the plates were washed with 1 mL of TE buffer, and the bacterial suspensions were processed for the identification of 24 species by DNA probes and the Checkerboard DNA-DNA hybridization method. Differences in total counts, prevalence, and levels of the species evaluated before and after placement of the strips were determined by Wilcoxon sign rank and Chi-square tests. RESULTS: A modest increase in the total bacterial number in saliva from 1.4 x 10(8) to 1.7 x 10(8) bacterial cells was observed 30 minutes after the strip placement, although this change was not significant (p = 0.632). Most of the species reduced in frequency and/or levels, including the pathogens A. actinomycetemcomitans, C. rectus, E. corrodens, Fusobacterium spp., P. intermedia, and S. noxia, as well as the beneficial species A. meyeri, A. georgia, A. gerencseriae, A. odontolyticus, and P. acnes after strip placement. In contrast, A. viscosus, P. melaninogenica, P. gingivalis, P. micros, Streptococcus spp., T. forsythensis, and V. parvula presented an increase in prevalence and/or levels. These changes were not statistically significant after adjusting for multiple comparisons (p > 0.0022). CONCLUSION: The use of the essential oil-containing oral strips resulted in a short-term small increase in the total number of salivary microorganisms. In addition, a not significant decrease of certain periodontopathogens, and an increase in species compatible with oral health were observed.

Susceptibility of oral bacteria to Melaleuca alternifolia (tea tree) oil in vitro.

The in vitro activity of Melaleuca alternifolia (tea tree) oil against 161 isolates of oral bacteria from 15 genera was determined. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) ranged from 0.003 to 2.0% (v/v). MIC90 values were 1.0% (v/v) for Actinomyces spp., Lactobacillus spp., Streptococcus mitis and Streptococcus sanguis, and 0.1% (v/v) for Prevotella spp. Isolates of Porphyromonas, Prevotella and Veillonella had the lowest MICs and MBCs, and isolates of Streptococcus, Fusobacterium and Lactobacillus had the highest. Time kill studies with Streptococcus mutans and Lactobacillus rhamnosus showed that treatment with > or = 0.5% tea tree oil caused decreases in viability of >3 log colony forming units/ml after only 30 s, and viable organisms were not detected after 5 min. These studies indicate that a range of oral bacteria are susceptible to tea tree oil, suggesting that tea tree oil may be of use in oral healthcare products and in the maintenance of oral hygiene.

Effect of an essential oil-containing dentifrice on dental plaque microbial composition.

PURPOSE: To determine the effect of 6 months use of an essential oil-containing (EO) antiplaque/antigingivitis fluoride dentifrice on the balance of the oral microbial flora and on the emergence of resistant microbial forms by analysis of dental plaque and saliva. MATERIALS AND METHODS: The dentifrice essential oils consisted of a fixed combination of thymol, menthol, methyl salicylate, and eucalyptol. An identical fluoride-containing dentifrice without the essential oils served as the control. A subgroup of 66 subjects from a clinical trial population of 321 was randomly selected for characterization of their dental plaque microflora. Saliva was also cultured to monitor for the emergence of opportunistic pathogens. Supragingival plaque and saliva were harvested at baseline, after which subjects received a dental prophylaxis. Subjects were sampled again after 3 and 6 months of product use prior to clinical examination. Plaque was characterized for microbial content by phase contrast microscopy for recognizable cellular morphotypes and by cultivation on nonselective and selective culture media. Determination of the minimum inhibitory concentrations of the test agent against selected Actinomyces and Veillonella isolated bacterial species was conducted at all time points to monitor for the potential development of bacterial resistance. RESULTS: There were no statistically significant differences between the microbial flora obtained from subjects using the essential oil-containing dentifrice and the vehicle control for all parameters and time periods except for the percentage of spirochetes at 6 months and for percentage of "other" microorganisms at 3 months. The EO group exhibited a lower adjusted mean for both parameters. Additionally, there was no evidence of the development of bacterial resistance to the antimicrobial activity of the essential oils or the emergence of opportunistic pathogens.

The effect of herbal extracts in an experimental mouthrinse on established plaque and gingivitis.

The purpose of the present study was to establish in vitro the inhibiting effect of a herbal extract mixture on a selected number of micro-organisms and to test in vivo the effect of a mouthwash containing 6.3 mg/ml herbal extract mixture on plaque and gingivitis as compared to a minus active control mouthrinse. The herbal extract was a mixture of: Juniperus communis (juniper), Urtica dioca (nettle), Achillaea millefolium (yarrow); 1:1:1. In the study, in-vitro, the effect of pure herbal extract mixture on acid production of Streptococcus mutans was tested and the minimum inhibitory concentrations (MIC) of the following micro-organisms were tested: Streptococcus mutans, Streptococcus mitis, Actynomyces viscosus, Actynomyces naeslundii, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, Veillonella parvula. The MIC-values for A. viscosus and P. gingivalis were 100 mg/ml. The MIC-values for A. naeslundii and A. actinomycetemcomitans were considerably lower (10 mg/ml). S. mitis was the most susceptible of the tested organisms to the extract with a MIC value of 1 mg/ml. S. mutans, C. rectus, V. parvula, and F. nucleatum were not influenced by the extracts. No inhibitory effect of the 6.3 mg/ml herbal extract mixture was observed on the acid production of S. mutans. For the study in-vivo, 45 volunteers were selected on the basis of having moderate gingival inflammation. As efficacy parameters the plaque index, modified gingival index and angulated bleeding index were assessed. The subjects were randomly divided among 3 experimental groups (2x test and 1 'minus active' control). The participants were requested to rinse with 10 ml of mouthwash twice a day for a period of three months. After 6 weeks and 3 months, the same clinical indices as at baseline were recorded. The results show no difference between the two test groups and the control group. In conclusion, the results of the present study have shown that the mixture of the 3 herbal extracts, Juniperus communis, Urtica dioca and Achillaea millefolium when used in a mouthrinse has no effect on plaque growth and gingival health.

Antimicrobial activity of magnolol and honokiol against periodontopathic microorganisms.

Magnolol (1) and honokiol (2), main compounds from the stem bark of Magnolia obovata Thunb., were evaluated for an antimicrobial activity against periodontopathic microorganisms, Porphyromonas gingivalis, Prevotella gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis, and Veillonella disper, and a cytotoxicity against human gingival fibroblasts and epithelial cells. Our results indicate that magnolol and honokiol, although less potent than chlorhexidine, show a significant antimicrobial activity against these microorganisms, and a relatively low cytotoxic effect on human gingival cells. Thus, it is suggested that magnolol and honokiol may have a potential therapeutic use as a safe oral antiseptic for the prevention and the treatment of periodontal disease.

Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents.

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.

A comparative study of cephradine, amoxycillin and phenoxymethylpenicillin in the treatment of acute dentoalveolar infection.

A comparative double blind trial was undertaken of the efficacy of cephradine, amoxycillin and phenoxymethylpenicillin in the treatment of acute dentoalveolar abscesses. Patients were admitted to the trial with acute dentoalveolar abscesses with systemic involvement and assessments were made of pain, swelling, temperature and lymphadenopathy. After admission to the trial appropriate surgical therapy was undertaken. The patients were then given either phenoxymethylpenicillin 250 mg four times daily, cephradine 500 mg twice daily or amoxycillin 250 mg three times daily, all for 5 days. The patients were assessed after 2 days and 5 days. All the patients recovered with those taking cephradine showing the best recovery in the first two days of treatment.

The effects of triclosan and zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro.

A mixed-culture chemostat system has been used as a more stringent laboratory system for evaluation of the antimicrobial effects of Triclosan and zinc citrate. The inhibitors were added alone, and in combination, as a pulse (a high initial inhibitor concentration which decreased with time) or as a dose (concentration of inhibitor increased with time) to give maximum concentrations of 34.5 mumol/L Triclosan and 39.8 mumol/L zinc citrate. When dosed, Triclosan inhibited A. viscosus and all five Gram-negative species, whereas zinc citrate had less effect, probably due to complexation by media components. Similar effects were seen when Triclosan was pulsed, except that S. mutans was the most sensitive Gram-positive species and V. dispar was unaffected. However, when the inhibitors were dosed or pulsed in combination, marked complementary and additive inhibitory effects were observed, particularly against Gram-negative species, although S. gordonii and S. oralis were relatively unaffected. The data confirm that increased effects can be obtained with suitable combinations of antimicrobial agents and suggest that, under certain conditions, apparently broad-spectrum antimicrobial agents may be acting more selectively than hitherto suspected.

Bacterial growth inhibition and short-term clinical efficacy of a vegetable oil-based mouthrinse: preliminary study.

Bacterial growth of a vegetable oil-based product was determined in vitro. Growth of Streptococcus mutans and Veillonella alcalescens was strongly inhibited for dilutions up to 50x. On the basis of these results, it was decided to evaluate the short-term, clinical efficacy of a 1:10 dilution of the concentrated product in water. Fifteen human volunteers employed this rinse twice-a-day during a six-day period. Planimetric plaque scores, as well as the plaque and gingival indexes according to Löe, were assessed at the beginning and end of this period. The efficacy of such a rinse, though less than of chlorhexidine-based products, turned out comparable to several other commercially available products with a reasonable potential in maintaining a low gingival index.

Erythromycin and anaerobic infection.

The theoretical basis of the use of erythromycin in peri-operative prophylaxis after bowel surgery will be discussed and the results of the use of erythromycin in such patients and the sensitivity to the antibiotic of isolates from human peritonitis described. This work will be related to results obtained in a rabbit experimental model for the study of peritonitis.

Effects of sodium trimetaphosphate supplementation of a high sucrose diet on the microbial and biochemical composition of four-day plaque and on urine calcium and phosphorus levels.

Daily supplementation of a high sucrose diet with 5 gm TMP resulted in significant alterations in certain human plaque components. Veillonella and Neisseria population (as percent total viable microorganisms), S sanguis (as percent streptococci), and levan hydrolase activity per milligram of plaque increased, whereas total viable microorganisms per milligram of plaque, streptococci per milligram of plaque, and specific fructosyltransferase activity decreased during the TMP-containing diet regimen.

Use of hydroxyapatite-coasted glass beads for preclinical testing of potential antiplaque agents.

Methods for rapid preclinical testing of antiplaque agents in vitro using hydroxyapatite (HT)-coated glass beads are described. The assays developed could reliably detect (i) prevention of growth in the culture fluid or on the HT surfaces, (ii) the effect of transient exposure of a bactericidal agent on the viability of cells in a preformed bacterial mat, (iii) reversible adsorption of a bactericidal agent on an HT surface, and (iv) the ability of an agent to inhibit adsorption of Streptococcus sanguis to an HT surface or to salivary proteins adsorbed to an HT surface.