Monika Astasov-Frauenhoffer

Microcalorimetric determination of the effects of amoxicillin, metronidazole, and their combination on in vitro biofilm.

BACKGROUND The mechanism of action of adjuvant antibiotic therapy in the treatment of peri-implantitis is not well understood. The aim of this study is to investigate antibiotic susceptibility of an in vitro biofilm by isothermal microcalorimetry (IMC). METHODS Titanium disks containing a 72-hour three-species biofilm (Streptococcus sanguinis DSM20068, Fusobacterium nucleatum ATCC10953, and Porphyromonas gingivalis DSM20709) were placed in a series of IMC ampoules with nutrient agar supplemented with increasing concentrations of amoxicillin, metronidazole, or their combination and incubated anaerobically for 10 days. Lag time and maximum growth rate were determined from continuous heat-flow recordings of metabolic activity. To validate the IMC biofilm results, adherent S. sanguinis and P. gingivalis were incubated anaerobically in medium supplemented with antibiotics at 37°C for 24 hours, and their vitality was determined by live/dead staining, conventional culturing, and IMC. RESULTS In all biofilm samples incubated with antibiotics, a prolonged lag phase was observed compared with controls (P <0.05). Maximum growth rate was significantly lower for samples treated with either amoxicillin or metronidazole compared with controls (P <0.05). Combining the antibiotics did not improve this effect. Concentrations exceeding 10 times the minimum inhibitory concentration completely inhibited the growth of adherent S. sanguinis and P. gingivalis, whereas lower concentrations resulted in only a delay in the lag phase. A poor correlation was observed between live/dead staining and conventional culturing. CONCLUSIONS IMC gives new evidence about antibiotic effects on oral biofilms and is more informative than conventional culture and live/dead assays. The combination of antibiotics was found to be more efficient than metronidazole alone; however, only minor differences in growth inhibition were detected compared with amoxicillin alone.

In Vitro Biofilm Formation on Titanium and Zirconia Implant Surfaces

BACKGROUND It has been hypothesized that zirconia might have a reduced bacterial adhesion compared with titanium; however, results from experimental studies are rather controversial. The aim of the present study is to compare biofilm formation on zirconia and titanium implant surfaces using an in vitro three-species biofilm and human plaque samples. METHODS Experimental disks made of titanium (Ti) or zirconia (ZrO2) with a machined (M) or a sandblasted (SLA) and acid-etched (ZLA) surface topography were produced. An in vitro three-species biofilm or human plaque samples were applied for bacterial adhesion to each type of disk, which after 72 hours of incubation was assessed using an anaerobic flow chamber model. RESULTS Zirconia showed a statistically significant reduction in three-species biofilm thickness compared with titanium (ZrO2-M: 8.41 μm; ZrO2-ZLA: 17.47 μm; Ti-M: 13.12 μm; Ti-SLA: 21.97 μm); however, no differences were found regarding three-species-biofilm mass and metabolism. Human plaque analysis showed optical density values of 0.06 and 0.08 for ZrO2-M and ZrO2-ZLA, and values of 0.1 and 0.13 for Ti-M and Ti-SLA, respectively; indicating a statistically significant reduction in human biofilm mass on zirconia compared with titanium. Additionally, zirconia revealed a statistically significant reduction in human plaque thickness (ZrO2-M: 9.04 μm; ZrO2-ZLA: 13.83 μm; Ti-M: 13.42 μm; Ti-SLA: 21.3 μm) but a similar human plaque metabolism compared with titanium. CONCLUSION Zirconia implant surfaces showed a statistically significant reduction in human plaque biofilm formation after 72 hours of incubation in an experimental anaerobic flow chamber model compared with titanium implant surfaces.

Bacterial colonization of resin composite cements: influence of material composition and surface roughness

So-called secondary caries may develop in the cement gap between the tooth and the bonded restoration. Cement materials with a low susceptibility to biofilm formation are therefore desirable. In the present study, the adhesion of Strepococcus mutans onto three adhesive (Multilink Automix, RelyX Ultimate, and Panavia V5) and three self-adhesive (Multilink Speed Cem, RelyX Unicem 2 Automix, and Panavia SA plus) resin composite cements was evaluated. Previous studies have failed to evaluate concomitantly the effect of both the composition of the cements and their surface roughness on biofilm formation. The presence of S. mutans on cement surfaces with differing degrees of roughness was therefore recorded using fluorescence microscopy and crystal violet staining, and the composition of the cements was analyzed using energy-dispersive X-ray spectroscopy mapping. Biofilm formation on resin composite cements was found to be higher on rougher surfaces, implying that adequate polishing of the cement gap is essential. The use of copper-containing cements (Multilink Automix, Panavia V5, and Panavia SA plus) significantly reduced biofilm formation.

Influence of the oscillation frequency of different side-to-side toothbrushes on noncontact biofilm removal

ObjectivesThe objective of this study was to investigate the influence of different oscillation frequencies of three powered toothbrushes with side-to-side action for noncontact biofilm removal in an artificial interdental space model.Materials and methodsA three-species biofilm (Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus sanguinis) was formed in vitro on protein-coated titanium disks using a flow chamber system combined with a static biofilm growth model. The oscillation frequencies of three commercial side-to-side toothbrushes were evaluated by means of a dose response. The frequency was decreased in steps (100, 85, 70, 55, and 40%). Subsequently, the biofilm-coated substrates were exposed to the side-to-side toothbrushes. The biofilm volumes were measured using volumetric analyses (Imaris 8.1.2) with confocal laser scanning microscope images (Zeiss LSM700).ResultsCompared to maximum oscillation frequency (100%), lower oscillation frequencies (up to 40%) resulted in reduced median percentages of biofilm reduction (median biofilm reduction up to 53% for maximum oscillation frequency, and up to 13% for 40% oscillation frequency) (p ≥ 0.03). In addition, decreasing the oscillation frequencies of the side-to-side toothbrushes showed an enhanced variety in the results of repeated experiments.ConclusionsThe oscillation frequency of the tested side-to-side toothbrushes affected the biofilm reduction in an interdental space model.Clinical relevanceWithin a toothbrush, higher oscillation frequencies may lead to beneficial effects on interdental biofilm removal by noncontact brushing.

Beneficial Oral Biofilms as Smart Bioactive Interfaces

Periodontitis is a very common health problem caused by formation of pathogenic bacterial biofilm that triggers inflammation resulting in either reversible gingivitis or irreversible periodontal hard and soft tissue damages, leading to loss of teeth when left untreated. Commensal bacteria play an important role in oral health in many aspects. Mainly by colonizing oral tissues, they (i) contribute to maturation of immune response, and (ii) foreclose attachment of pathobiont and, therefore, prevent from infection. The main goal of the study was to investigate if blocking of receptors on a commensal biofilm can prevent or reduce the attachment of pathogenic strains. To do so, biofilm produced by commensal Streptococcus sanguinis was treated with whole cell lysate of pathobionts Fusobacterium nucleatum or Porphyromonas gingivalis, followed by incubation with respective strain(s). The study revealed significant reduction in pathobiont adhesion to lysate-treated commensal biofilm. Therefore, adhesion of pathobionts onto the lysate-blocked biofilm was hindered; however, not completely eliminated supporting the idea that such approach in the oral cavity would benefit the production of a well-balanced and healthy bioactive interface.

Biofilm formation on restorative materials and resin composite cements

OBJECTIVES Monolithic zirconia, polymer-infiltrated ceramic and acrylate polymer cemented with resin composite cement have recently been identified as prosthetic treatment options for zirconia implants. The aim of the present study is to determine in vitro, to what extent bacteria adhere to these materials. METHODS Disks made of zirconia (Vita YZ [YZ]), polymer-infiltrated ceramic (Vita Enamic [VE]), acrylate polymer (Vita CAD-Temp [CT]), self-adhesive cement (RelyX Unicem 2 Automix [RUN]) and of two different adhesive cements (RelyX Ulimate [RUL] and Vita Adiva F-Cem [VAF]) were produced. The biofilm formation of three bacterial species (Streptococcus sanguinis, Fusobacterium nucleatum, Porphyromonas gingivalis) on each material was assessed over 72h using a flow chamber system. The biofilms were quantified by crystal violet staining (optical density 595nm) and visualized using SEM. The inorganic composition of the different materials was analyzed and the wettability of the specimens was measured. RESULTS For the restorative materials lowest biofilm formation was found on CT: OD 0.5±0.1, followed by VE: OD 0.8±0.1 and YZ: OD 1.4±0.3. The biofilm formation on resin composite cements was significantly lower on VAF: OD 0.6±0.1 than for RUL: OD 0.9±0.1 and RUN: OD 1.0±0.1. A high wettability of the specimens with saliva/serum mixture tended to result in a higher biofilm formation. Correlations were obtained between the organic/inorganic composition of the materials and the polar/dispersive part of the surface free energy. SIGNIFICANCE Three-species biofilm formation on restorative and cement materials strongly relies on the materials composition. If the restorative material CT and cement VAF also prevent excessive biofilm formation in a clinical situation should be further investigated.

Influence of the amplitude of different side-to-side toothbrushes on noncontact biofilm removal

ObjectivesTo investigate the impact of the lateral deflection of toothbrush bristles (amplitude) of three side-to-side toothbrushes for noncontact biofilm removal in an artificial interdental space model.Materials and methodsA three-species biofilm (Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus sanguinis) was formed in vitro on protein-coated titanium disks. A flow chamber system was combined with a static biofilm growth model. The amplitudes of three commercial side-to-side toothbrushes were evaluated by means of a dose response analysis. The amplitudes were decreased in steps (100%, 85%, 70%, 55%, and 40%). Subsequently, the biofilm-coated substrates were exposed to the toothbrushes. The biofilms were analyzed with confocal laser scanning microscope images and measured using volumetric analyses.ResultsThe predictability of interdental biofilm reduction differed among the toothbrushes. A lower variety in the results of repeated experiments occurred in toothbrush C compared to toothbrushes A and B. Toothbrush C obtained highest percentage of biofilm reduction by 85% of amplitude power setting (median biofilm reduction 76%). Decreasing the amplitude from 85 to 40% resulted in reduced biofilm reduction (p = 0.029). In contrast, no significance could be observed for the differences of the tested amplitudes within toothbrushes A and B (p > 0.05). Between the toothbrushes, a significant difference in interdental biofilm reduction was found between C-A (p = 0.029) and C-B (p = 0.029) with amplitude of 85%.ConclusionsThe amplitude of one of the investigated side-to-side toothbrushes affected the biofilm reduction predictably in an interdental space model.Clinical relevanceWithin certain toothbrushes, a specific amplitude power setting may demonstrate beneficial effects on noncontact biofilm removal.

Exopolysaccharides regulate calcium flow in cariogenic biofilms

Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.

Antibacterial effects of bio-inspired nanostructured materials

ABSTRACT Several properties of bio-inspired surfaces like chemical composition, surface topography, surface hydrophilicity and even surface charge could influence bacterial adhesion to implant materials. Therefore, a nanostructured surface is being investigated to avoid bacterial colonization by their physico-mechanical and chemical aspects. Both smooth and rough-surfaced titanium (PT, SLA) and zirconia (M and ZLA) surfaces were used as controls. Titanium SLA was modified by two-step-etching to create nanostructured surface. Antibacterial properties of the materials were tested by adhesion of Porphyromonas gingivalis (ATCC 33277). The vitality of bacteria was assessed by Live/Dead BacLight™ Bacterial Viability Kit or by conventional culturing on Columbia blood agar. Conventional culturing revealed reduction of bacteria on nanostructured titanium (5.27±0.8 x 104 CFU/mm2) in comparison to rough-surfaced control materials (ZLA 6.16±4.86 x 104 and SLA 1.53±0.75 x 105 CFU/mm2). However, smooth-surfaced control materials (M 2.25±0.84 x 104 and PT 6.63±5.77 x 103 CFU/mm2) showed similar results to the nanostructured material. Live/dead staining demonstrated the antimicrobial efficacy of the nanostructured material revealing reduction of vital bacteria population up to 70%. This effect was not observed on the control materials (bacterial vitality ≥95%). In conclusion, nanostructured titanium surface shows a reduction of vital bacteria. Therefore, bio-inspired nanostructures can modify the bacteria–titanium interaction.