Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose.

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n = 306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

Microbial approaches for the assessment of toothpaste efficacy against oral species: A method comparison.

Antibacterial properties of toothpastes enable chemical plaque control in limited-access tooth regions that are mechanically not sufficiently reached by toothbrushes. Therefore, this study aimed to compare different microbial methods to assess antimicrobial toothpaste properties and evaluate different toothpastes in terms of their antibacterial efficacy against different oral microorganisms in an in vitro setting. Six toothpaste suspensions with varying antibacterial supplements were applied to a multispecies biofilm model (Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, and Streptococcus mutans) as well as to each microorganism. A culture method was used to assess the anti-biofilm effects and two different agar diffusion assays were performed for testing the antimicrobial effect on each microorganism. The measurements of the culture and diffusion analyses were statistically normalized and compared and toothpastes were ranked according to their antimicrobial efficacy. The results of both agar diffusion assays showed a high correlation across all tested species (Spearman correlation coefficients ρs > 0.95). The results of the multispecies biofilm model, however, substantially differed in its assessment of antibacterial properties (ρs ranging from 0.22 to 0.87) compared to the results of both diffusion assays. Toothpastes with amine fluoride (with and without stannous fluoride), and toothpastes with triclosan resulted in the highest antimicrobial efficacy. Activated carbon supplements in toothpastes were comparable in their antimicrobial action to the negative control NaCl. The appropriate selection of a broad range of oral microorganisms seems crucial when testing the chemical impact of toothpaste and toothpaste supplements.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Salivary Biomarkers for Dental Caries Detection and Personalized Monitoring.

This study investigated the potential of salivary bacterial and protein markers for evaluating the disease status in healthy individuals or patients with gingivitis or caries. Saliva samples from caries- and gingivitis-free individuals (n = 18), patients with gingivitis (n = 17), or patients with deep caries lesions (n = 38) were collected and analyzed for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). The resulting data were subjected to principal component analysis and used as a training set for random forest (RF) modeling. This computational analysis revealed four biomarkers (IL-4, IL-13, IL-2-RA, and eotaxin/CCL11) to be of high importance for the correct depiction of caries in 37 of 38 patients. The RF model was then used to classify 10 subjects (five caries-/gingivitis-free and five with caries), who were followed over a period of six months. The results were compared to the clinical assessments of dental specialists, revealing a high correlation between the RF prediction and the clinical classification. Due to the superior sensitivity of the RF model, there was a divergence in the prediction of two caries and four caries-/gingivitis-free subjects. These findings suggest IL-4, IL-13, IL-2-RA, and eotaxin/CCL11 as potential salivary biomarkers for identifying noninvasive caries. Furthermore, we suggest a potential association between JAK/STAT signaling and dental caries onset and progression.

Single DNase or Proteinase Treatment Induces Change in Composition and Structural Integrity of Multispecies Oral Biofilms.

Biofilm virulence is mainly based on its bacterial cell surrounding biofilm matrix, which contains a scaffold of exopolysaccharides, carbohydrates, proteins, lipids, and nucleic acids. Targeting these nucleid acids or proteins could enable an efficient biofilm control. Therefore, the study aimed to test the effect of deoxyribonuclease I (DNase I) and proteinase K on oral biofilms. Six-species biofilms (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Fusobacterium nucleatum, Veillonella dispar, and Candida albicans) were exposed to DNase I (0.001 mg/mL, 0.002 mg/mL) or proteinase K (0.05 mg/mL, 0.1 mg/mL) for 1 h during biofilm formation. After 64 h, biofilms were harvested, quantified by culture analysis and visualized by image analysis using CLSM (confocal laser scanning microscopy). Statistical analysis was performed by ANOVA, followed by the Tukey test at a 5% significance level. The biofilm treatment with proteinase K induced a significant increase of Logs10 counts in S. mutans and a decrease in C. albicans, while biofilm thickness was reduced from 28.5 µm (control) to 9.07 µm (0.05 mg/mL) and 7.4 µm (0.1 mg/mL). Treatment with DNase I had no effect on the total bacterial growth within the biofilm. Targeting proteins of biofilms by proteinase K are promising adjunctive tool for biofilm control.

Staphylococcus aureus Interferes with Streptococci Spatial Distribution and with Protein Expression of Species within a Polymicrobial Oral Biofilm.

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases.

Toothpastes with Enzymes Support Gum Health and Reduce Plaque Formation.

Enzymes in toothpastes can support host immune responses, and thus maintain oral health. This study aimed to investigate gingival health and the plaque-reducing effects of enzyme-containing toothpastes. A laboratory study tested the antimicrobial potential of different enzyme-containing toothpaste formulations. Two promising formulations (enzyme-containing toothpastes with glucose oxidase and D-glucose with (C+) and without Citrox (C-) Citrox) were investigated in a clinical crossover trial (two slurries: sodium lauryl sulfate-containing (SLS), a toothpaste without SLS (reference), and water). Subjects (n = 20) abstained from toothbrushing for four days and rinsed with a toothpaste slurry. Bleeding on probing (BOP) and plaque indices (PI) were measured. A mixed linear model was used to statistically compare the slurries with respect to BOP and PI change. The in vitro bacterial growth-inhibiting evaluation showed the best results for SLS, followed by C+ and C-. The change in BOP and PI exhibited statistically significant differences to water rinsing (BOP; PI changes in % points (difference of the baseline and post-rinse values: water = 8.8%; 90.0%; C+ = -1.4%; 80.4%; SLS = 1.5%; 72.1%; reference = 0.8%; 77.5%; C- = -1.8%; 75.1%). All slurries exhibited anti-gingivitis and anti-plaque effects, resulting in a prophylactic benefit for limited-access regions during brushing.

Microbial Analysis of Saliva to Identify Oral Diseases Using a Point-of-Care Compatible qPCR Assay.

Oral health is maintained by a healthy microbiome, which can be monitored by state-of-the art diagnostics. Therefore, this study evaluated the presence and quantity of ten oral disease-associated taxa (P. gingivalis, T. forsythia, T. denticola, F. nucleatum, C. rectus, P. intermedia, A. actinomycetemcomitans, S. mutans, S. sobrinus, oral associated Lactobacilli) in saliva and their clinical status association in 214 individuals. Upon clinical examination, study subjects were grouped into healthy, caries and periodontitis and their saliva was collected. A highly specific point-of-care compatible dual color qPCR assay was developed and used to study the above-mentioned bacteria of interest in the collected saliva. Assay performance was compared to a commercially available microbial reference test. Eight out of ten taxa that were investigated during this study were strong discriminators between the periodontitis and healthy groups: C. rectus, T. forsythia, P. gingivalis, S. mutans, F. nucleatum, T. denticola, P. intermedia and oral Lactobacilli (p < 0.05). Significant differentiation between the periodontitis and caries group microbiome was only shown for S. mutans (p < 0.05). A clear distinction between oral health and disease was enabled by the analysis of quantitative qPCR data of target taxa levels in saliva.

Impact of interdental brush shape on interpapillary cleaning efficacy - a clinical trial.

This study aimed to investigate whether interdental brush shape influences cleaning efficacy, by comparing a waist-shaped interdental brush (W-IDB) with a cylindrical IDB (C-IDB); both provided with the same bristle texture. Cleaning efficacy of differently shaped IDBs was measured in proximal surfaces of teeth in a split-mouth cross-over design. Twenty-eight patients abolished oral hygiene for 4 d. Line angle plaque area was scanned with an intraoral camera after use of disclosing dye in baseline and after IDB application and analyzed planimetrically. Additionally, bacterial load in the IDBs was analyzed after usage by colony forming units (cfu). A Wilcoxon signed-rank test with continuity correction was used to compare the results of the waist-shaped and the cylindrically-shaped IDBs. The waist-shaped IDBs cleaned significantly better than their cylindrically-shaped counterparts (area cleaned: 23.1% vs. 18.3%), when applied at same interdental spaces (p < 0.001). However, no significant differences were found in comparison of bacterial load on the IDBs (median cfu counts: 2.3E9 vs. 2.7E9, p = 0.93). Irrespective of bristle texture or size, IDB shape have impact on cleaning efficacy. Waist-shaped IDBs are more effective in cleaning of the line angle area than cylindrically-shaped IDBs.