Optimizing the use of low-frequency ultrasound for bacterial detachment of in vivo biofilms in dental research-a methodological study.

OBJECTIVES: Low-frequency, low-intensity ultrasound is commonly utilized in various dental research fields to remove biofilms from surfaces, but no clear recommendation exists in dental studies so far. Therefore, this study aims to optimize the sonication procedure for the dental field to efficiently detach bacteria while preserving viability. MATERIALS AND METHODS: Initial biofilm was formed in vivo on bovine enamel slabs (n = 6) which were worn by four healthy participants for 4 h and 24 h. The enamel slabs covered with biofilm were then ultrasonicated ex vivo for various time periods (0, 1, 2, 4, 6 min). Colony-forming units were determined for quantification, and bacteria were identified using MALDI-TOF. Scanning electron microscopic images were taken to also examine the efficiency of ultrasonications for different time periods. RESULTS: Ultrasonication for 1 min resulted in the highest bacterial counts, with at least 4.5-fold number compared to the non-sonicated control (p < 0.05). Most bacteria were detached within the first 2 min of sonication, but there were still bacteria detached afterwards, although significantly fewer (p < 0.0001). The highest bacterial diversity was observed after 1 and 2 min of sonication (p < 0.03). Longer sonication periods negatively affected bacterial counts of anaerobes, Gram-negative bacteria, and bacilli. Scanning electron microscopic images demonstrated the ability of ultrasound to desorb microorganisms, as well as revealing cell damage and remaining bacteria. CONCLUSIONS: With the use of low-frequency, low-intensity ultrasound, significantly higher bacterial counts and diversity can be reached. A shorter sonication time of 1 min shows the best results overall. CLINICAL RELEVANCE: This standardization is recommended to study initial oral biofilms aged up to 24 h to maximize the outcome of experiments and lead to better comparability of studies.

Improving the microbial sampling and analysis of secondary infected root canals by passive ultrasonic irrigation.

OBJECTIVE: The persistence of pathogenic microorganisms in root canals is the most common reason for the failure of root canal treatment and the necessity of a root filling treatment, which results in an uncertain prognosis due to technical complexity and the variety of highly adaptable microorganisms. This study evaluated the effect of passive ultrasonic irrigation (PUI) on the outcome of the microbial analysis of root canal-treated teeth with persistent or recurrent apical inflammation in vivo. MATERIALS AND METHODS: Sample collection was performed after root filling removal (sample S1, control group) and after PUI with NaCl (sample S2) using sterile paper points. In total, 19 samples were obtained. Quantification was performed by means of serial dilution of the samples. Subcultivated pure cultures were identified using MALDI-TOF MS complemented by the Vitek-2-System or PCR, followed by sequencing of the 16S rRNA gene. The results of the samples (S1 and S2) were evaluated regarding their bacterial count and composition. RESULTS: The total count of bacteria and the number of aerobic/facultative anaerobic microorganisms significantly increased in the S2-samples after application of PUI. The number of obligate anaerobic microorganisms showed an increase after PUI, although it was not significant. We detected 12 different aerobic/facultative anaerobic microorganisms before PUI, and in 21 cases after PUI. Two different obligate anaerobic microorganisms were found in S1 samples compared to nine different species in S2 samples. CONCLUSIONS: PUI is a powerful method for detaching bacteria in infected root canals and enables a more precise analysis of the etiology of persistent endodontic infections. CLINICAL RELEVANCE: This study indicates that PUI exerts a positive cleansing effect and adds to the accessibility of microorganisms during the application of bactericidal rinsing solution in root canal treatments.

Colonization of Helicobacter pylori in the oral cavity - an endless controversy?

Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori. Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori. Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori-like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.

Limited antimicrobial efficacy of oral care antiseptics in microcosm biofilms and phenotypic adaptation of bacteria upon repeated exposure.

OBJECTIVES: The aims of this study were to investigate the antimicrobial efficacy of antiseptics in saliva-derived microcosm biofilms, and to examine phenotypic adaption of bacteria upon repeated exposure to sub-inhibitory antiseptic concentrations. METHODS: Saliva-derived biofilms were formed mimicking caries- or gingivitis-associated conditions, respectively. Microbial compositions were analyzed by semiconductor-based 16S rRNA sequencing. Biofilms were treated with CHX, CPC, BAC, ALX, and DQC for 1 or 10 min, and colony forming units (CFU) were evaluated. Phenotypic adaptation of six selected bacterial reference strains toward CHX, CPC, and BAC was assessed by measuring minimum inhibitory concentrations (MICs) over 10 passages of sub-inhibitory exposure. Protein expression profiles were investigated by SDS-PAGE. RESULTS: Both biofilms showed outgrowth of streptococci and Veillonella spp., while gingivitis biofilms also showed increased relative abundances of Actinomyces, Granulicatella, and Gemella spp. Antiseptic treatment for 1 min led to no relevant CFU-reductions despite for CPC. When treated for 10 min, CPC was most effective followed by BAC, ALX, CHX, and DQC. Stable adaptations with up to fourfold MIC increases were found in E. coli toward all tested antiseptics, in E. faecalis toward CHX and BAC, and in S. aureus toward CPC. Adapted E. coli strains showed different protein expression as compared with the wildtype strain. CONCLUSION: Antiseptics showed limited antimicrobial efficacy toward mature biofilms when applied for clinically relevant treatment periods. Bacteria showed phenotypic adaptation upon repeated sub-inhibitory exposure. CLINICAL RELEVANCE: Clinicians should be aware that wide-spread use of antiseptics may pose the risk of inducing resistances in oral bacteria.

In vivo and in vitro analysis in a rat model using zoledronate and alendronate medication: microbiological and scanning electron microscopy findings on peri-implant rat tissue.

BACKGROUND: The aim of the present study was to assess the development of bacterial deposits and morphological parameters around dental zirconia and titanium implants compared with natural teeth during systemic bisphosphonate medication. MATERIALS AND METHODS: Fifty-four rats were randomly allocated into one control group and two experimental groups (drug application of zoledronic and alendronic acid), with 18 animals in each group. After 4 weeks of drug delivery, either a zirconia or a titanium implant was immediately inserted. Microbiological analysis conducted 1 week, 8 weeks, and 12 weeks after surgery included total bacterial count and composition measurements. Samples were analyzed in a scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDX). Bone cell morphology was analyzed by transmission electron microscopy (TEM). RESULTS: One week after surgery, titanium and zirconia implants of the alendronic acid and control group showed a significantly higher bacterial count when compared to natural teeth in rats with zoledronic acid administration (p < 0.01). Less significant differences were recorded after 3 months, at which time no inter-material differences were evaluated (p > 0.05). I n the control group, TEM analysis showed that the osteoblasts had a strongly developed endoplasmic reticulum. In contrast, the endoplasmic reticulum of the osteoblasts in drug-treated animals was significantly less developed, indicating less activity. CONCLUSIONS: Within the limits of this study, neither implant material was superior to the other at 3-month follow-up. With regard to the treatment and complications of patients with bisphosphonates, the implant material should not be an influencing factor. Bisphosphonates can be used in the rat model to reduce not only the activity of osteoclasts but also osteoblasts of the peri-implant bone.

Cetylpyridinium Chloride: Mechanism of Action, Antimicrobial Efficacy in Biofilms, and Potential Risks of Resistance.

Antimicrobial resistance is a serious issue for public health care all over the world. While resistance toward antibiotics has attracted strong interest among researchers and the general public over the last 2 decades, the directly related problem of resistance toward antiseptics and biocides has been somewhat left untended. In the field of dentistry, antiseptics are routinely used in professional care, but they are also included in lots of oral care products such as mouthwashes or dentifrices, which are easily available for consumers over-the-counter. Despite this fact, there is little awareness among the dental community about potential risks of the widespread, unreflected, and potentially even needless use of antiseptics in oral care. Cetylpyridinium chloride (CPC), a quaternary ammonium compound, which was first described in 1939, is one of the most commonly used antiseptics in oral care products and included in a wide range of over-the-counter products such as mouthwashes and dentifrices. The aim of the present review is to summarize the current literature on CPC, particularly focusing on its mechanism of action, its antimicrobial efficacy toward biofilms, and on potential risks of resistance toward this antiseptic as well as underlying mechanisms. Furthermore, this work aims to raise awareness among the dental community about the risk of resistance toward antiseptics in general.

Long-Term Fluctuation of Oral Biofilm Microbiota following Different Dietary Phases.

Caries development is associated with shifts in the oral biofilm microbiota and primarily linked to frequent simple carbohydrate consumption. Different nutritional ingredients can either promote or prevent caries development. To investigate the effects of selected ingredients on the oral biofilm microbiota in situ, 11 study participants underwent 3-month-long dietary phases with intake of a regular diet (PI), additional frequent sucrose (PII), milk and yoghurt (PIII), and a diet rich in dietary fiber (PIV) and then returned to their regular diet (PV). Oral biofilm was sampled and analyzed applying 16S rRNA Illumina MiSeq sequencing. Additionally, the effect on the enamel was analyzed by measuring enamel surface roughness with laser scanning microscopy. The beta-diversity results showed that the microbiota in all the following phases differed significantly from PI and that the microbial community in PII was significantly different from all other phases. The abundance of the genus Streptococcus fluctuated over the course of the five phases, with a significant increase in PII (P = 0.01), decreasing in PIII and PIV (PIII and PIV versus PII: P < 0.00001) and increasing again toward PV. Other taxa showed various fluctuations of their abundances, with PV returning approximately to the levels of PI. In conclusion, while elevated sucrose consumption favored caries-promoting non-mutans streptococci, frequent milk and yoghurt intake caused a significant decrease in the abundance of these microbial taxa and in addition reduced enamel surface roughness. These results indicate that modulations of the oral biofilm microbiota can be attained even in adults through dietary changes and corresponding recommendations can be made for the prevention of caries development.IMPORTANCE Caries affects a large proportion of the population worldwide, resulting in high treatment costs. Its etiology can be ascribed to shifts of the microbiota in dental biofilms primarily driven by dietary factors. It is unclear how diet affects the microbial community of plaque biofilm in situ and whether it can be modulated to help prevent caries development. To address these issues, we analyzed changes of the in situ plaque microbiota following 3-month-long dietary changes involving elevated sucrose, dairy, and dietary fiber consumption over a period of 15 months. Applying high-throughput sequencing, we found non-mutans streptococci, a taxonomic group involved in the beginning stages toward microbial dysbiosis, in decreased abundance with elevated dairy and dietary fiber intake. Through analysis of the enamel surface roughness, these effects were confirmed. Therefore, correspondent dietary measures can be recommended for children as well as adults for caries prevention.

In vitro evaluation of the cariogenic potential of rebaudioside A compared to sucrose and xylitol.

OBJECTIVES: The aim of the present study was to evaluate the growth and acid synthesis of cariogenic pathogens in vitro when rebaudioside A was the sole source of carbon, compared to sucrose, xylitol, and a commercial sweetener containing rebaudioside A. MATERIALS AND METHODS: First, sucrose or sugar substitutes were added to human saliva and the pH was measured over 10 h. Subsequently, the growth and acid synthesis efficiency of Streptococcus mutans, Streptococcus sobrinus, Streptococcus oralis, Lactobacillus rhamnosus, Lactobacillus paracasei, and Candida albicans were investigated: after adding them to minimal culture media containing only one of the carbon sources, the optical density and pH were determined over 10 h. RESULTS: A significant pH drop only occurred in the sucrose group of human saliva whereas no differences were found between the sugar substitute groups. The individual strains of caries pathogens showed significantly lower growth rates and less acid synthesis in the sugar replacement groups, in comparison to sucrose. However, these groups also showed bacterial growth and a decline in pH. CONCLUSION: Xylitol appeared to be superior to rebaudioside A regarding the inhibition of mutans streptococci. The results for the remaining microorganisms indicate that rebaudioside A causes a similar or lower acid synthesis than xylitol. As saliva contains buffer systems, the metabolism of rebaudioside A did not seem to shift the equilibrium to demineralization. CLINICAL RELEVANCE: Rebaudioside A may not be suitable as a targeted Streptococcus mutans treatment, but it could contribute to a reduction of caries risk as a replacement for sucrose.

Effects of subgingival air-polishing with trehalose powder on oral biofilm during periodontal maintenance therapy: a randomized-controlled pilot study.

BACKGROUND: This pilot study was part of a larger study which compared the effect of subgingival air-polishing using trehalose powder with sonic scaling on clinical parameters during supportive periodontal therapy. Within this microbiological part of the investigation subgingival samples were taken from 10 participants to analyze the survival of different bacterial species after the two different treatments as a proof of principle. METHODS: In 10 participants two non-adjacent, single-root teeth requiring treatment (PD =5 mm with bleeding on probing (BOP) or > 5 mm) were selected following a split-mouth design and were treated either with a sonic scaler or air-polishing device and trehalose powder. For persistent pockets (PD =4 mm and BOP or > 4 mm), treatment was repeated after 3 months. Subgingival biofilm samples were taken at baseline (BL), subsequently and three and six months after treatment. After determination of the bacterial counts (TBL), isolated bacteria were identified by MALDI-TOF-MS. If unsuccessful, PCR and 16S rDNA sequencing were performed. RESULTS: In both treatment groups, TBL decreased immediately after treatment remaining at a lower level. This confirms the findings of the larger study regarding clinical parameters showing a comparable effect on PD, BOP and CAL. Immediately after treatment, the diversity of detected species decreased significantly more than in the sonic group (p = 0.03). After 3 months, the proportion of Gram-positive anaerobic rods was lower in the air-polishing group (powder/ sonic 7%/ 25.9%, p = 0.025). Also, there was a greater reduction of Gram-negative aerobic rods for this group at this time (air-polishing/ sonic - 0.91 / -0.23 Log10 cfu/ ml, p = 0.020). CONCLUSION: Within the limitations of this study air-polishing and sonic treatment seem to have a comparable effect on the subgingival oral biofilm during supportive periodontal treatment. TRIAL REGISTRATION: The study was registered in an international trial register (German Clinical Trial Register number DRKS 00006296) on 10th of June 2015. HTML&TRIAL_ID = DRKS00006296.

Three-Dimensional, Bifunctional Microstructured Polymer Hydrogels Made from Polyzwitterions and Antimicrobial Polymers.

Biofilm-associated infections of medical devices are a global problem. For the prevention of such infections, biomaterial surfaces are chemically or topographically modified to slow down the initial stages of biofilm formation. In the bifunctional material here presented, chemical and topographical cues are combined, so that protein and bacterial adhesion as well as bacterial proliferation are effectively inhibited. Upon changes in the surface topography parameters and investigation of the effect of these changes on bioactivity, structure-property relationships are obtained. The target material is obtained by microcontact printing (µCP), a soft lithography method. The antimicrobial component, poly(oxanorbornene)-based synthetic mimics of an antimicrobial peptide (SMAMP), was printed onto a protein-repellent polysulfobetaine hydrogel, so that bifunctional 3D structured polymer surfaces with 1, 2, and 8.5 µm spacing are obtained. These surfaces are characterized with fluorescence microscopy, surface plasmon resonance spectroscopy, atomic force microscopy, and contact angle measurements. Biological studies show that the bifunctional surfaces with 1 and 2 µm spacing are 100% antimicrobially active against Escherichia coli and Staphylococcus aureus, 100% fibrinogen-repellent, and nontoxic to human gingival mucosal keratinocytes. At 8.5 µm spacing, the broad-band antimicrobial activity and the protein repellency are compromised, which indicates that this spacing is above the upper limit for effective simultaneous antimicrobial activity and protein repellency of polyzwitterionic-polycationic materials.

The influence of an anti-inflammatory diet on gingivitis. A randomized controlled trial.

AIM: Aim of this study was to investigate the influence of an anti-inflammatory diet on different parameters in patients with gingivitis. MATERIALS AND METHODS: Thirty patients were randomly allocated to an experimental and a control group stratified by their plaque values. The experimental group had to change to a diet low in processed carbohydrates and animal proteins, and rich in omega-3 fatty acids, vitamin C, vitamin D, antioxidants, plant nitrates and fibres for 4 weeks. The control group did not change their diet. Both groups suspended interdental cleaning. Periodontal parameters were assessed by a blinded dentist. Serological and subgingival plaque samples were taken at baseline and end. RESULTS: While there were no differences regarding the plaque values, the experimental group showed a significant reduction in gingival bleeding (GI Baseline: 1.04 ± 0.21, GI End: 0.61 ± 0.29, p < 0.05), a significant increase in Vitamin D values and a significant weight loss. There were no inter-group differences regarding the inflammatory serological parameters, the serological omega fatty acids, nor the subgingival microbiome composition. CONCLUSION: The evaluated diet could significantly reduce gingivitis in a clinically relevant range, while serological inflammatory parameters and the subgingival microbiome seem to be unaffected in this study duration. (German Clinical Trials Register; DRKS00009888).

Compounds from Olea europaea and Pistacia lentiscus inhibit oral microbial growth.

BACKGROUND: In view of the increasing antibiotic resistance, the introduction of natural anti-infective agents has brought a new era in the treatment of bacterially derived oral diseases. METHODS: The aim of this study was to investigate the antimicrobial potential of five natural constituents of Olea europaea (oleuropein, maslinic acid, hydroxytyrosol, oleocanthal, oleacein) and three compounds of Pistacia lentiscus (24Z-isomasticadienolic acid, oleanolic acid, oleanonic aldehyde) against ten representative oral bacterial species and a Candida albicans strain. After the isolation and quality control of natural compounds, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay were performed. RESULTS: Among all O. europaea-derived constituents, maslinic acid was the most active (MIC = 4.9-312 µg mL- 1, MBC = 9.8-25 µg mL- 1) one against oral streptococci and anaerobic pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra), while oleuropein, hydroxytyrosol, oleocanthal and oleacein showed milder, yet significant effects against P. gingivalis and F. nucleatum. Among all P. lentiscus compounds, oleanolic acid was the most effective one against almost all microorganisms with MIC values ranging from 9.8 µg mL- 1 (P. gingivalis) to 625 µg mL- 1 (F. nucleatum, P. micra). In the presence of 24Z-isomasticadienolic acid, a mean inhibitory concentration range of 2.4 µg mL- 1 to 625 µg mL- 1 was observed for strict anaerobia. The MIC value for 24Z-isomasticadienolic acid was estimated between 39 µg mL- 1 (Streptococcus sobrinus, Streptococcus oralis) and 78 µg mL- 1 (Streptococcus mutans). All tested compounds showed no effects against Prevotella intermedia. CONCLUSIONS: Overall, maslinic acid and oleanolic acid exerted the most significant inhibitory activity against the tested oral pathogens, especially streptococci and anaerobic oral microorganisms.

Antibiotic Resistance Genes and Antibiotic Susceptibility of Oral Enterococcus faecalis Isolates Compared to Isolates from Hospitalized Patients and Food.

Enterococcus faecalis, a commensal of the intestinal tract of humans and animals is of great significance as leading opportunistic pathogen, and also prevalent in oral diseases, such as endodontic infections, as well as the healthy oral cavity. To investigate the potential of oral E. faecalis to constitute a reservoir of antibiotic resistance, isolates from supragingival plaque/saliva and from endodontic infections were screened regarding their resistance to selected antibiotics in comparison to nosocomial and food isolates.70 E. faecalis isolates were analyzed with PCR regarding their equipment with the resistance genes tetM, tetO, ermB, ermC, vanA, vanB and blaTEM. Additionally, they were tested for their phenotypic resistance to doxycycline, azithromycin, rifampicin, amoxicillin and streptomycin using the Etest.High percentages of the plaque/saliva, nosocomial and food isolates were resistant to doxycycline and azithromycin, particularly plaque/saliva isolates (81%) and nosocomial isolates (73.3%) showed resistance to doxycycline, significantly more than among the food and endodontic isolates. Rifampicin resistance was widespread among isolates from plaque/saliva (52.4%), endodontic infections (50%) and nosocomial infections (40%); all isolates were susceptible to amoxicillin and all oral isolates to high-level streptomycin. TetM genes were detected in the majority of all isolates and ermB genes were present in many nosocomial and plaque/saliva isolates. Thirty percent of the endodontic isolates and 53% of the nosocomial isolates were equipped with blaTEM genes.The results suggest that the oral cavity can harbor E. faecalis strains with multiple resistances against different antibiotics and thus be regarded as a potential source of resistance traits.

Activity of preparations from Spilanthes oleracea, propolis, Nigella sativa, and black garlic on different microorganisms involved in oral diseases and on total human salivary bacteria: A pilot study.

Due to continuous rise in antibiotic resistance, there is a need for alternative treatment options to reduce the levels of oral pathogens for the maintenance of oral as well as overall health. The aim of this study was to evaluate the in vitro antibacterial potential of tinctures of Spilanthes oleracea and propolis, Nigella seed oil, and an ethanolic extract of black garlic on microorganisms involved in oral diseases. Both the minimum inhibitory concentration assay and the minimum bactericidal/fungicidal concentration assay were used in this study. Inhibition effects against total human salivary bacteria were also determined. Our results show that all of the preparations tested had potent antimicrobial activities. When measured 10 min after exposure, even low concentrations of the propolis tincture were found to have killed more than 99% of salivary bacteria, whereas Spilanthes tincture and black garlic extract killed more than 90% and Nigella seed oil more than 60% of the pathogens. This suggests that all preparations are promising candidates for the use in oral health care products and that all have the potential to control biofilm associated infections.

Association between high risk for preterm birth and changes in gingiva parameters during pregnancy-a prospective cohort study.

OBJECTIVES: The objective of this study was to investigate clinical and microbiological gingival changes during pregnancy in women without periodontal disease. Additionally, these parameters were to be compared in women with high risk for preterm birth and women with a normal course of pregnancy. METHOD AND MATERIALS: Group I consisted of 40 subjects at high risk for preterm birth, while group II involved 49 subjects with a normal course of pregnancy. The control group (III) was made up of 50 non-pregnant women. Clinical parameters (plaque index, gingival index, probing pocket depths, gingival swelling, bleeding on probing) and microbiological changes were monitored during pregnancy and 2-4 weeks after parturition. RESULTS: In the high-risk preterm group (I), 19 women could be included in data analysis. This group was compared to 41 women in the normal pregnancy group (II) and 50 non-pregnant women (III). Gingival inflammation was significantly higher in women with high risk for preterm birth (I) compared to non-risk pregnant women (II, p < 0.05). In addition, in this group (I), the subgingival amounts of Fusobacterium nucleatum (> 105) were found to be significantly higher after childbirth compared to non-pregnant women (p < 0.05). CONCLUSIONS: Even without having periodontal disease, women with high risk for preterm birth showed worse clinical values compared to non-risk pregnant and non-pregnant women and an increased detection of Fusobacterium nucleatum after delivery. CLINICAL RELEVANCE: High risk for preterm birth might be associated with the occurrence of increased gingival inflammation.

A Simultaneously Antimicrobial, Protein-Repellent, and Cell-Compatible Polyzwitterion Network.

A simultaneously antimicrobial, protein-repellent, and cell-compatible surface-attached polymer network is reported, which reduces the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based zwitterion (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass, and gold and real-life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the PZI surface and the two reference surfaces SMAMP ("synthetic mimic of an antimicrobial peptide"), an antimicrobial but protein-adhesive polymer coating, and PSB (poly(sulfobetaine)), a protein-repellent but not antimicrobial polyzwitterion coating were characterized by Fourier transform infrared spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy, swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy. The time-dependent antimicrobial activity assay (time-kill assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of Escherichia coli and Staphylococcus aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the live-dead stain, and the hemolysis assay. PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

Effect of gaseous ozone on Enterococcus faecalis biofilm-an in vitro study.

OBJECTIVES: The aim of this study was to evaluate the antimicrobial effect of gaseous ozone compared to conventional methods against Enterococcus faecalis. MATERIALS AND METHODS: One hundred twenty-five teeth were infected by E. faecalis and were incubated for 72 h to form biofilm. Teeth were distributed among five groups. In the first group, ozone was used; in the second group, teeth were rinsed with 20 % ethylenediaminetetraacetic acid (EDTA); in the third group, with 3 % sodium hypochlorite (NaOCl). Group 4 combined 20 % EDTA with ozone. NaOCl and ozone were combined in group 5. After treatment, the samples with paper points were taken, followed by dentin samples taken with K-file, and cultured for 24 h. Then bacterial colonies were counted. RESULTS: All treatments reduced significantly (p < 0.05) the bacteria. Paper points' samples showed 85.38 % reduction after ozone. The highest reduction was observed in NaOCl group (99.98 %). EDTA reduced bacteria by 80.64 %. Combination of NaOCl and ozone eradicated 99.95 % of the bacteria. Combination of EDTA and ozone reduced E. faecalis up to 91.33 %. The dentin chips showed the following: the highest CFU counts were observed in EDTA group, followed by ozone and NaOCl group. The lowest CFU counts were found in NaOCl-ozone group and EDTA-ozone group. CONCLUSIONS: Ozone reduced E. faecalis, even organised in a biofilm, however, lower than NaOCl. No treatment reduced totally the bacteria. CLINICAL RELEVANCE: Used as an adjuvant, ozone can increase the efficacy of conventional rinsing like EDTA and presents an alternative treatment when NaOCl cannot be used e.g. in teeth with a wide-open apical foramen.

Microbial adhesion on novel yttria-stabilized tetragonal zirconia (Y-TZP) implant surfaces with nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) coatings.

OBJECTIVES: Biomaterial surfaces are at high risk for initial microbial colonization, persistence, and concomitant infection. The rationale of this study was to assess the initial adhesion on novel implant surfaces of Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans upon incubation. MATERIALS AND METHODS: The tested samples were 3 mol% yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP) samples with nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) coating (A) and 3Y-TZP samples coated with ceria-stabilized zirconia-based (Ce-TZP) composite and a-C:H:N (B). Uncoated 3Y-TZP samples (C) and bovine enamel slabs (BES) served as controls. Once the surface was characterized, the adherent microorganisms were quantified by estimating the colony-forming units (CFUs). Microbial vitality was assessed by live/dead staining, and microbial-biomaterial surface topography was visualized by scanning electron microscopy (SEM). RESULTS: Overall, A and B presented the lowest CFU values for all microorganisms, while C sheltered significantly less E. faecalis, P. aeruginosa, and C. albicans than BES. Compared to the controls, B demonstrated the lowest vitality values for E. coli (54.12 %) and C. albicans (67.99 %). Interestingly, A (29.24 %) exhibited higher eradication rates for S. aureus than B (13.95 %). CONCLUSIONS: Within the limitations of this study, a-C:H:N-coated 3Y-TZP surfaces tended to harbor less initially adherent microorganisms and selectively interfered with their vitality. CLINICAL RELEVANCE: This could enable further investigation of the new multi-functional zirconia surfaces to confirm their favorable antimicrobial properties in vivo.

The use of computer-assisted FACE for minimal-invasive caries excavation.

OBJECTIVES: Caries excavation is still a demanding procedure today. In the past years, methods using light-induced fluorescence have proven their ability to detect bacteria remaining in the tooth's hard tissues. However, methods to control the complete removal of infected dentine have not yet been validated. The aim of the present study was to evaluate the degree of dentine removal during caries excavation using a camera- and software-based device as a guide for fluorescence-aided caries excavation (FACE) in comparison to visual-tactile inspection and a dye-staining method. MATERIALS AND METHODS: One hundred teeth with carious lesions (C3) were randomly allocated to four groups and excavated with the respective method as primary excavation control method. When the first method indicated complete caries excavation, a second method was used to affirm this. Subsequently, dentine samples were taken to determine residual contamination using PCR. After embedding and sectioning the teeth dentine, microhardness was measured perpendicular to the cavity floor. RESULTS: By using the camera- and software-based device, 94 % of the cavities tested were free of bacterial contamination. All cavities excavated with the dye-staining or visual-tactile control method were free of bacterial contamination but resulted in a significantly higher dentine removal. CONCLUSION: The camera- and software-assisted caries excavation seems to be an adequate method to remove contaminated dentine without removing affected dentine. CLINICAL RELEVANCE: Beside its original application to monitor carious, the device is safe, effective, and easy to use for guided caries excavation and is of worth for both everyday practice and undergraduate education.

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.