Antibacterial effect of taurolidine (2%) on established dental plaque biofilm

Preliminary data have suggested that taurolidine may bear promising disinfectant properties for the therapy of bacterial infections. However, at present, the potential antibacterial effect of taurolidine on the supragingival plaque biofilm is unknown. To evaluate the antibacterial effect of taurolidine on the supragingival plaque biofilm using the vital fluorescence technique and to compare it with the effect of NaCl and chlorhexidine (CHX), 18 subjects had to refrain from all mechanical and chemical hygiene measures for 24 h. A voluminous supragingival plaque sample was taken from the buccal surfaces of the lower molars and wiped on an objective slide. The sample was then divided into three equal parts and mounted with one of the three test or control preparations (a) NaCl, (b) taurolidine 2% and (c) CHX 0.2%. After a reaction time of 2 min, the test solutions were sucked of. Subsequently, the plaque biofilm was stained with fluorescence dye and vitality of the plaque flora was evaluated under the fluorescence microscope (VF%). Plaque samples treated with NaCl showed a mean VF of 82.42 ± 6.04%. Taurolidine affected mean VF with 47.57 ± 16.60% significantly (p < 0.001, paired t test). The positive control CHX showed the lowest mean VF values (34.41 ± 14.79%; p < 0.001 compared to NaCl, p = 0.017 compared to taurolidine). Taurolidine possesses a significant antibacterial effect on the supragingival plaque biofilm which was, however, not as pronounced as that of CHX.

Microbiota in the oral subgingival biofilm is associated with obesity in adolescence

To test the hypothesis whether microbiota in oral biofilm is linked with obesity in adolescents we designed this cross-sectional study. Obese adolescents (n = 29) with a mean age of 14.7 years and normal weight subjects (n = 58) matched by age and gender were examined with respect to visible plaque index (VPI%) and gingival inflammation (bleeding on probing (BOP%)). Stimulated saliva was collected. They answered a questionnaire concerning medical history, medication, oral hygiene habits, smoking habits, and sociodemographic background. Microbiological samples taken from the gingival crevice was analyzed by checkerboard DNA-DNA hybridization technique. The sum of bacterial cells in subgingival biofilm was significantly associated with obesity (P < 0.001). The link between sum of bacterial cells and obesity was not confounded by any of the studied variables (chronic disease, medication, VPI%, BOP%, flow rate of whole saliva, or meal frequency). Totally 23 bacterial species were present in approximately threefold higher amounts, on average, in obese subjects compared with normal weight controls. Of the Proteobacteria phylum, Campylobacter rectus and Neisseria mucosa were present in sixfold higher amounts among obese subjects. The association between obesity and sum of bacterial cells in oral subgingival biofilm indicates a possible link between oral microbiota and obesity in adolescents.

Dendritic glycopeptides as Pseudomonas aeruginosa biofilm inhibitors, Oral presentation, 32nd European Peptide symposium, Athens, Greece, 2-7 September 2012

Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors. Glycopeptide dendrimers are being developed for inhibition of pathogen adhesion to host cells, a process mediated by carbohydrate-lectins interactions. Such compounds could be used in the treatment of infections by pathogenic bacteria such as Pseudomonas aeruginosa that can be resistant to known antibiotics. Pseudomonas aeruginosa produces two lectins, the fucose binding LecB and the galactose binding LecA. Both lectins have been shown to be virulence factors, involved in cell adhesion and biofilms formation. Screening combinatorial libraries of fucosylated peptide dendrimers led to the glycopeptide dendrimer (C-Fuc-LysProLeu)4(LysPheLysIle)2 LysHisIleNH2. This dendrimer binds the lectin LecB with submicromolar IC50 and shows potent inhibition of P. aeruginosa biofilms for both the laboratory strain PAO1 and for clinical isolates [1]. Appending the peptide dendrimer portion of FD2 with galactosy endgroups gave galactosylpeptide dendrimers as potent ligands for LecA which also act as biofilm inhibitors. Structure-activity relationship studies demonstrated that multivalency was essential for strong binding and biofilm inhibition. [2]The results open the way to develop therapeutic agents based on glycopeptide dendrimers. Peptide dendrimers with antimicrobial properties and good cell penetration are other applications of dendritic peptides we are now investigating.

Biofilm-related infections of cerebrospinal fluid shunts

Cerebrospinal fluid (CSF) shunts carry a high risk of complications. Infections represent a major cause of shunt failure. Diagnosis and therapy of such infections are complicated by the formation of bacterial biofilms attached to shunt surfaces. This study correlated the pathophysiology and clinical course of biofilm infections with microscopical findings on the respective shunts. Surface irregularities, an important risk-factor for shunt colonisation with bacteria, were found to increase over time because of silicone degradation. Scanning electron-microscopy (SEM) documented residual biological material (dead biofilm), which can further promote extant bacterial adhesion, on newly manufactured shunts. Clinical course and SEM both documented bacterial dissemination against CSF flow and the monodirectional valve. In all cases, biofilms grew on both the inner and outer surfaces of the shunts. Microscopy and conventional culture detected all bacterial shunt infections. Analyses of 16S rDNA sequences using conserved primers identified bacteria in only one of three cases, probably because of previous formalin fixation of the samples.

Clinical effects of interdental cleansing on supragingival biofilm formation and development of experimental gingivitis

PURPOSE: The aim of the present study was to test the effects of interdental cleansing with dental floss on supragingival biofilm removal in natural dentition during a 3-week period of experimental biofilm accumulation. MATERIALS AND METHODS: The present study was performed as a single-blind, parallel, randomised, controlled clinical trial using the experimental gingivitis model (Löe et al, 1965). Thirty-two students were recruited and assigned to one of the following experimental or control groups: Group A used a fluoride-containing dentifrice (NaF dentifrice) on a toothbrush for 60 s twice a day, Group B used an unwaxed dental floss twice a day, Group C used a waxed dental floss twice a day in every interproximal space and Group D rinsed twice a day for 60 s with drinking water (control). RESULTS: During 21 days of abolished oral hygiene, the groups developed various amounts of plaque and gingivitis. Neither of the cleansing protocols alone allowed the prevention of gingivitis development. Toothbrushing alone yielded better outcomes than did any of the flossing protocols. Interdental cleansing with a waxed floss had better biofilm removal effects than with unwaxed floss. CONCLUSIONS: Toothbrushing without interdental cleansing using dental floss and interdental cleansing alone cannot prevent the development of gingivitis.

A paradigm shift in mechanical biofilm management? Subgingival air polishing: a new way to improve mechanical biofilm management in the dental practice

In the past few years indications for the use of the air polishing technology have been expanded from supragingival use (airflow) to subgingival air polishing (perioflow) by the development of new low-abrasive glycine-based powders and devices with a subgingival nozzle. Several studies on the subgingival use of air polishing have been completed. On 7 June 2012, during the Europerio 7 Congress in Vienna, a consensus conference on mechanical biofilm management took place aiming to review the current evidence from the literature on the clinical relevance of the subgingival use of air polishing and to make practical recommendations for the clinician. Bernita Bush (Bern), Prof Johannes Einwag (Stuttgart), Prof Thomas Flemmig (Seattle), Carmen Lanoway (Munich), Prof Ursula Platzer (Hamburg), Prof Petra Schmage (Hamburg), Brigitte Schoeneich (Zurich), Prof Anton Sculean (Bern), Dr Clemens Walter (Basel), and Prof Jan Wennström (Gothenburg) discussed under the moderation of Klaus-Dieter Bastendorf and Christian Becker (both ADIC Association for Dental Infection Control) the available clinical studies to reach a consensus on available clinical evidence. This paper summarizes the main conclusions of the consensus conference and points to the clinical relevance of the findings for the dental practitioner.

Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm

The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria associated with periodontitis within a biofilm (single strain and mixed population) in vitro. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed populations in a planktonic state. Single-species biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multi-species biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the antibacterials (0.002 - 512 µg ml-1) for 18 h, addition of nutrient broth for 3 days and subsequent subcultivation. Photographs were taken by using confocal laser scanning microscopy and scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin against A. actinomycetemcomitans, moxifloxacin was more active than the other tested antibacterials against anaerobes and the mixed population. The single-species biofilms were eradicated by moderate concentrations of the antibacterials, the lowest MBECs were always found for moxifloxacin (2-8 µg ml-1). MBECs against the multi-species biofilms were 128 µg ml-1, >512 µg ml-1 and >512 µg ml-1 for moxifloxacin, ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the biofilms.

The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

Action of food preservatives on 14-days dental biofilm formation, biofilm vitality and biofilm-derived enamel demineralisation in situ

AIMS The aims of this double-blind, controlled, crossover study were to assess the influence of food preservatives on in situ dental biofilm growth and vitality, and to evaluate their influence on the ability of dental biofilm to demineralize underlying enamel over a period of 14 days. MATERIALS AND METHODS Twenty volunteers wore appliances with six specimens each of bovine enamel to build up intra-oral biofilms. During four test cycles of 14 days, the subjects had to place the appliance in one of the assigned controls or active solutions twice a day for a minute: negative control 0.9 % saline, 0.1 % benzoate (BA), 0.1 % sorbate (SA) and 0.2 % chlorhexidine (CHX positive control). After 14 days, the biofilms on two of the slabs were stained to visualize vital and dead bacteria to assess biofilm thickness (BT) and bacterial vitality (BV). Further, slabs were taken to determine mineral loss (ML), by quantitative light-induced laser fluorescence (QLF) and transversal microradiography (TMR), moreover the lesion depths (LD). RESULTS Nineteen subjects completed all test cycles. Use of SA, BA and CHX resulted in a significantly reduced BV compared to NaCl (p < 0.001). Only CHX exerted a statistically significant retardation in BT as compared to saline. Differences between SA and BA were not significant (p > 0.05) for both parameters. TMR analysis revealed the highest LD values in the NaCl group (43.6 ± 44.2 μm) and the lowest with CHX (11.7 ± 39.4 μm), while SA (22.9 ± 45.2 μm) and BA (21.4 ± 38.5 μm) lay in between. Similarly for ML, the highest mean values of 128.1 ± 207.3 vol% μm were assessed for NaCl, the lowest for CHX (-16.8 ± 284.2 vol% μm), while SA and BA led to values of 83.2 ± 150.9 and 98.4 ± 191.2 vol% μm, respectively. With QLF for both controls, NaCl (-33.8 ± 101.3 mm(2) %) and CHX (-16.9 ± 69.9 mm(2) %), negative values were recorded reflecting a diminution of fluorescence, while positive values were found with SA (33.9 ± 158.2 mm(2) %) and BA (24.8 ± 118.0 mm(2) %) depicting a fluorescence gain. These differences were non-significant (p > 0.05). CONCLUSION The biofilm model permited the assessment of undisturbed oral biofilm formation influenced by antibacterial components under clinical conditions for a period of 14 days. An effect of BA and SA on the demineralization of enamel could be demonstrated by TMR and QLF, but these new findings have to be seen as a trend. As part of our daily diet, these preservatives exert an impact on the metabolism of the dental biofilm, and therefore may even influence demineralization processes of the underlying dental enamel in situ.

Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation, Surface Alterations and Attachment of Periodontal Ligament Fibroblasts.

BACKGROUND AND AIM There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD. RESULTS After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts. CONCLUSION The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.

Multivalency effects in Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA

The galactose specific lectin LecA partly mediates the formation of antibiotic resistant biofilms by Pseudomonas aeruginosa, an opportunistic pathogen causing lethal airways infections in immunocompromised and cystic fibrosis patients, suggesting that preventing LecA binding to natural saccharides might provide new opportunities for treatment. Here 8-fold (G3) and 16-fold (G4) galactosylated analogs of GalAG2, a tetravalent G2 glycopeptide dendrimer LecA ligand and P. aeruginosa biofilm inhibitor, were obtained by convergent chloroacetyl thioether (ClAc) ligation between 4-fold or 8-fold chloroacetylated dendrimer cores and digalactosylated dendritic arms. Hemagglutination inhibition, isothermal titration calorimetry and biofilm inhibition assays showed that G3 dendrimers bind LecA slightly better than their parent G2 dendrimers and induce complete biofilm inhibition and dispersal of P. aeruginosa biofilms, while G4 dendrimers show reduced binding and no biofilm inhibition. A binding model accounting for the observed saturation of glycopeptide dendrimer galactosyl groups and LecA binding sites is proposed based on the crystal structure of a G3 dendrimer LecA complex.