Subgingival Biofilm Research Articles

Extracellular host DNA contributes to pathogenic biofilm formation during periodontitis.

Periodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis. In this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides-Porphyromonas-Prevotella (BPP) group associated with periodontitis. Analysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis. eDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

Step 2 for the treatment of periodontal diseases: adjunctive therapies

Step 2 of periodontal therapy primarily aims at the non-surgical, mechanical disruption of the subgingival biofilm and removal of plaque-retentive factors, allowing a shift from disease to health. Numerous therapeutic agents have been proposed as adjuncts to non-surgical periodontal therapy, generally through either aiding disruption of the dental plaque biofilm, or though alteration of the host immune response. This article reviews the British Society of Periodontology's implementation of the S3-level guidelines in relation to adjunctive therapies. CPD/Clinical Relevance: Recommendation to use adjunctive therapies in the treatment of periodontitis requires careful consideration of the available evidence as well as the wider effects of the proposed therapy.

Step 2 for the treatment of periodontal diseases

This article discusses the modalities of treatment involved in step 2 of periodontal therapy, including exploring looking at the evidence-based recommendations produced by the S3 stages I–III periodontal treatment guideline. CPD/Clinical Relevance: Step 2 of periodontal therapy is essential for removal of the subgingival biofilm and reduction in the bacterial load.

Preliminary study on the diversity and quantity analysis of oral bacteria according to the sampling methods

Objectives: Oral bacterial samples included subgingival, supragingival, and saliva plaques. As the diversity and number of microorganisms deffer depending on the area of the oral cavity and the method used, an appropriate and reliable collection method is important. The present study investigated oral bacterial sampling methods. Methods: Supragingival dental plaque was collected from the buccal and lingual tooth surfaces of study participants using sterilized cotton swabs. Plaques were collected from the subgingival area using a sterilized curette. Bacterial genomic DNA was extracted using MagNA Pure 96 DNA and Viral NA low-volume kits. Real-time polymerase chain reaction (PCR) was performed using the PowerCheck™ Periodontitis Pathogens Multiplex Real-time PCR kit. Results: Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum of the orange complex were not observed in the subgingival biofilms of all study participants. For Porphyromonas. gingivalis, a significant correlation was observed between supragingival, subgingival, and total tooth surface biofilms. Compared to the supragingival and subgingival biofilmss, total tooth surface biofilm exhibited the highest bacterial count when the inswabbing method was used. Conclusions: Based on these findings, the supragingival swab method is recommended for oral bacterial research.

Erythritol powder airflow for the treatment of peri-implant mucositis: A randomized controlled clinical trial.

Peri-implant mucositis is a biofilm-related, reversible inflammatory disease that can evolve into peri-implantitis if not adequately treated. The aim of the present randomized controlled clinical trial was to evaluate the efficacy of air-abrasive powder as compared to chlorhexidine (CHX) for the treatment of peri-implant mucositis, in terms of clinical and patient-reported outcomes (PROMs) and occurrence of peri-implantitis 12 months after treatment. In the control group, full-mouth calculus and plaque removal was performed with ultrasound and manual devices, and a 1.0% CHX gel was applied; in the test group, supra- and subgingival biofilm removal was performed using erythritol powder with a dedicated nozzle and calculus removal was performed with ultrasonic instruments if needed. Bleeding and plaque indexes, peri-implant probing depth and tissue level were measured at 1 week, and 1, 3, 6 and 12 months after treatment, while PROMs were evaluated up to 7 days after treatment. Among 80 included implants, 70 were analysed at 12 months follow-up (30 in the test group, 40 in the control group, and 20 subjects). Success rates (implant-level) in terms of bleeding index were significantly different between the test (96.7%) and control group (92.5%); as for PROMs, only taste sensation was significantly better in the test group. The test group was significantly correlated to the smallest changes in peri-implant probing depth between baseline and 3 months. The study showed that both treatment strategies are effective. This suggests that the use of air-abrasive powders could be used as an alternative biofilm removal method instead of adjunctive treatments with antiseptics.

Antimicrobial Activity of Methylene Blue Associated with Photodynamic Therapy: In Vitro Study in Multi-Species Oral Biofilm.

The control of infectious diseases caused by biofilms is a continuing challenge for researchers due to the complexity of their microbial structures and therapeutic implications. Photodynamic therapy as an adjunctive anti-infective treatment has been described as a possible valid approach but has not been tested in polymicrobial biofilm models. This study evaluated the effect of photodynamic therapy in vitro with methylene blue (MB) 0.01% and red LEDs (λ = 660 nm, power density ≈ 330 mW/cm2, 2 mm distance from culture) on the metabolic activity and composition of a multispecies subgingival biofilm. Test Groups LED and MB + LED showed a more significant reduction in metabolic activity than the non-LED application group (~50 and 55%, respectively). Groups LED and MB equally affected (more than 80%) the total bacterial count in biofilms. No differences were noted in the bacterial biofilm composition between the groups. In vitro LED alone or the MB + LED combination reduced the metabolic activity of bacteria in polymicrobial biofilms and the total subgingival biofilm count.

Antibiofilm and Immune-Modulatory Activity of Cannabidiol and Cannabigerol in Oral Environments-In Vitro Study.

To evaluate the in vitro antimicrobial and antibiofilm properties and the immune modulatory activity of cannabidiol (CBD) and cannabigerol (CBG) on oral bacteria and periodontal ligament fibroblasts (PLF). Cytotoxicity was assessed by propidium iodide flow cytometry on fibroblasts derived from the periodontal ligament. The minimum inhibitory concentration (MIC) of CBD and CBG for S. mutans and C. albicans and the metabolic activity of a subgingival 33-species biofilm under CBD and CBG treatments were determined. The Quantification of cytokines was performed using the LEGENDplex kit (BioLegend, Ref 740930, San Diego, CA, USA). CBD-treated cell viability was greater than 95%, and for CBG, it was higher than 88%. MIC for S. mutans with CBD was 20 µM, and 10 µM for CBG. For C. albicans, no inhibitory effect was observed. Multispecies biofilm metabolic activity was reduced by 50.38% with CBD at 125 µg/mL (p = 0.03) and 39.9% with CBG at 62 µg/mL (p = 0.023). CBD exposure at 500 µg/mL reduced the metabolic activity of the formed biofilm by 15.41%, but CBG did not have an effect. CBG at 10 µM caused considerable production of anti-inflammatory mediators such as TGF-β and IL-4 at 12 h. CBD at 10 µM to 20 µM produced the highest amount of IFN-γ. Both CBG and CBD inhibit S. mutans; they also moderately lower the metabolic activity of multispecies biofilms that form; however, CBD had an effect on biofilms that had already developed. This, together with the production of anti-inflammatory mediators and the maintenance of the viability of mammalian cells from the oral cavity, make these substances promising for clinical use and should be taken into account for future studies.

Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples

BackgroundSequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively.ResultsWith both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing.ConclusionsThe results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

Oxygen enrichment mediated by calcium peroxide loaded gelatin methacrylate hydrogel eradicates periodontal biofilms

The low oxygen environment of the periodontal pocket favors pathogenic anaerobes' growth, biofilm formation, and quick recurrence after periodontal treatment. In contrast, oxygen is detrimental to anaerobes, such as Porphyromonas gingivalis (P. gingivalis), since they lack a complete anti-oxidation mechanism to detoxify the oxygen challenge. Therefore, consistently feeding pathogenic anaerobes with abundant oxygen would be an effective strategy to combat them. Here, we reported injectable oxygen-generating hydrogels as oxygen mediators to alleviate the local anaerobic environment and eliminate periodontal pathogens. Gelatin methacrylate (GelMA) hydrogels loaded with calcium peroxide (CPO) possessed excellent injectability and exhibited burst releases of oxygen within 24 h with a 40 % oxygen tension peak. CPO-GelMA hydrogels with CPO concentrations of 5, 10, and 15 % reduced 60, 99, and 89.9 % viable P. gingivalis, respectively. Five percentage CPO-GelMA hydrogel downregulated gingipain and fimA gene expression in P. gingivalis without resistance development. Moreover, the CPO-GelMA hydrogels remarkably prevented biofilm formation and eradicated both monospecies and multispecies bacterial biofilms. In conclusion, CPO-GelMA hydrogels exert remarkable antimicrobial and antibiofilm effects on subgingival biofilms, providing a promising strategy for periodontal treatment.

Antibacterial effect of green tea against Porphyromonas gingivalis: a literature review

Green tea is regarded as a healthy beverage due to the biological action of polyphenols, specifically catechins. Researchers investigated the inhibitory effects of green tea (Camellia sinensis) plant on both Gram- positive and Gram-negative bacteria. Periodontitis is mainly initiated when dysbiosis of the subgingival microbiome takes place. Porphyromonas gingivalis is one of the key pathogens involved in the initiation and progression of periodontitis when its levels in subgingival biofilm overwhelm the host's immune system. It is the main pathogen that is significantly linked to severe periodontal disease. In this study, details about P. gingivalis including information about its structure, virulence factors were provided. Next, Important points related to green tea, including its active ingredients and antibacterial activity, have been clarified. Finally, the objective of this study was to demonstrate how green tea has antibacterial effects on P. gingivalis.

The Impact of Candida albicans in the Development, Kinetics, Structure, and Cell Viability of Biofilms on Implant Surfaces-An In Vitro Study with a Validated Multispecies Biofilm Model.

This study aimed to evaluate the impact of Candida albicans on subgingival biofilm formation on dental implant surfaces. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to compare biofilm structure and microbial biomass in the presence and absence of the fungus after periods of 24, 48, and 72 h. Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total micro-organisms for each of the biofilm-forming strains. A general linear model was applied to compare CLSM and qPCR results between the control and test conditions. The biofilm developed with C. albicans at 72 h had a higher bacterial biomass and a significantly higher cell viability (p < 0.05). After both 48 and 72 h of incubation, in the presence of C. albicans, there was a significant increase in counts of Fusobacterium nucleatum and Porphyromonas gingivalis and in the cell viability of Streptococcus oralis, Aggregatibacter actinomycetemcomitans, F. nucleatum, and P. gingivalis. Using a dynamic in vitro multispecies biofilm model, C. albicans exacerbated the development of the biofilm grown on dental implant surfaces, significantly increasing the number and cell viability of periodontal bacteria.

Evaluation of microbiome in primary and permanent dentition in grade C periodontitis in young individuals.

The aim of the present study was to evaluate the subgingival microbiome in patients with grade C molar-incisor pattern periodontitis (C-MIP) affecting the primary or permanent dentitions. DNA was isolated from subgingival biofilm samples from diseased and healthy sites from 45 C-MIP patients and subjected to phylogenetic microarray analysis. C-MIP sites were compared between children affected in the primary to those affected in the permanent dentitions. Within-subject differences between C-MIP-affected sites and dentition-matched healthy sites were also evaluated. C-MIP sites of subjects affected in the primary dentition showed partially overlapping but distinct microbial communities from C-MIP permanent dentition sites (p<0.05). Differences were due to increased levels in primary C-MIP sites of certain species of the genera Capnocytophaga and Leptotrichia, while C-MIP permanent dentition sites showed higher prevalence of Filifactor alocis. Aggregatibacter actinomycetemcomitans (Aa) was among species seen in high prevalence and levels in both primary and permanent C-MIP sites. Moreover, both permanent and primary C-MIP sites showed distinct microbial communities when compared to dentition-matched healthy sites in the same subject (p<0.01). Primary and permanent teeth with C-MIP showed a dysbiotic microbiome, with children affected in the primary dentition showing a distinct profile from those affected in the permanent dentition. However, Aa was enriched in both primary and permanent diseased sites, confirming that this microorganism is implicated in C-MIP in both dentitions.

Subgingival biofilm microbiome in individuals with asthma and periodontitis: Metagenomic analysis.

This observational study aimed to explore the metagenomics of subgingival biofilms in individuals with varying degrees of asthma, from severe to none, to elucidate the association between the subgingival microbiome and asthma. Subgingival biofilm samples were collected from thirty participants at the Asthma Control Program Outpatient Clinic in Bahia (ProAR). These samples were categorized into six groups based on the severity of asthma and the presence or absence of periodontitis. We employed next-generation sequencing (Illumina MiSeq), targeting the 16S rRNA gene, to characterize the microbial communities present. Our analysis included descriptive statistics and sequencing data, evaluated using multivariate statistical methods such as the Shannon index, principal coordinate analysis, and the Bray-Curtis dissimilarity. Our findings indicate a higher prevalence of periodontally detrimental bacterial genera in individuals with severe asthma and periodontitis. Additionally, individuals with asthma, but without periodontitis, exhibited a tendency toward dysbiosis, particularly in cases of severe asthma. This research provides new insights into the composition of the subgingival microbiome in individuals with varying severities of asthma and periodontitis. The genera identified in this study underscore the need for further investigations to build upon these findings.

MANUFACTURING A NEW MEDICATED CHEWABLE GUM FROM NATURAL MATERIALS TO CONTROL PERIODONTAL PATHOGENS

Within their multispecies subgingival biofilm in deep pockets, periodontal pathogens are being protected from the attendance of antimicrobial agents, in addition to; the licensed safe concentrations of antibacterials are less effective against biofilm; which leads to unsuccessful treatment and recurrent infections. Therefore, as a first local effort, the medical aim of the present study was to apply the natural material, frankincense, to make a medical chewing gum and an intra-pocket antimicrobial delivery system as a hopeful solution for the ineffectiveness of antimicrobials to control periodontal pathogens. The ability of frankincense film to work as an antibiotic delivery tool was tested by agar diffusion procedure against three types of anaerobic pathogens isolated from periodontal pockets. The usefulness of the currently prepared medical chewing gum from frankincense and other natural additives to control periodontal infections was tested by measuring the level of bacterial hydrolytic enzymes in gingival pockets. The results proved the validity of frankincense film to be a natural delivery tool of antimicrobial ingredients by the denotation of inhibiting the growth on agar plate. The results also proved the validity of frankincense to formulate a medical chewable gum with profound efficiency in reducing the bacterial load inside the gingival tissues. The study claims for the practical application of frankincense in the field of medicines production as a gum base to produce a medical chewable gum and an antimicrobial delivery system. This application can be adopted by pharmaceutical factories and can be dependable by the Ministry of Health and dentists as an alternative to increasing the concentration or combination of chemical medicines

Effectiveness of Ultrasonic and Manual Instrumentation in Nonsurgical Periodontal Therapy: Are Additional Therapies More Effective? A Systematic Review

Nonsurgical periodontal therapy aims to remove supragingival and subgingival biofilm to restore periodontal health. This systematic review aims to assess the clinical effectiveness of manual and/or ultrasonic instruments and to determine whether other therapies can improve periodontal clinical outcomes. Case-control, cross-sectional and cohort studies and clinical trials of patients undergoing nonsurgical periodontal therapy with ultrasonic and/or manual instruments (and any adjunctive therapies, such as glycine, erythritol, ozone, laser and glycine) from 2013 to 2023 were analyzed using Pub-med/MEDLINE, Scopus and Google Scholar. To assess the risk of bias in this review, blinding, randomization, allocation concealment, outcome data and outcome recording were assessed. No differences between treatments were found; all methods, including manual and ultrasound, were helpful in improving clinical parameters (primary outcome). Although the results were mixed, adjunctive therapies seemed to be helpful in the treatment of periodontal disease. The results of this systematic review are consistent with the previous scientific literature and have shown that both manual and ultrasonic instruments are effective in nonsurgical periodontal therapy. This review could not show how complementary therapies could further improve nonsurgical periodontal therapy. For future research, it would be good to standardize the sample with regard to the degree and stage of periodontal disease and to evaluate the risks and benefits of the instruments (manual and ultrasonic scalers).

Next-generation antibacterial nanopolymers for treating oral chronic inflammatory diseases of bacterial origin.

'Periodontitis' refers to periodontal destruction of connective tissue attachment and bone, in response to microorganisms forming subgingival biofilms on the root surface, while 'apical periodontitis' refers to periapical inflammatory processes occurring in response to microorganisms within the root canal system. The treatment of both diseases is based on the elimination of the bacterial challenge, though its predictability depends on the ability of disrupting these biofilms, what may need adjunctive antibacterial strategies, such as the next-generation antibacterial strategies (NGAS). From all the newly developed NGAS, the use of polymeric nanotechnology may pose a potential effective approach. Although some of these strategies have only been tested invitro and in preclinical invivo models, their use holds a great potential, and therefore, it is relevant to understand their mechanism of action and evaluate their scientific evidence of efficacy. To explore NGAS based on polymeric nanotechnology used for the potential treatment of periodontitis and apical periodontitis. A systemic search of scientific publications of adjunctive antimicrobial strategies using nanopolymers to treat periodontal and periapical diseases was conducted using The National Library of Medicine (MEDLINE by PubMed), The Cochrane Oral Health Group Trials Register, EMBASE and Web of Science. Different polymeric nanoparticles, nanofibres and nanostructured hydrogels combined with antimicrobial substances have been identified in the periodontal literature, being the most commonly used nanopolymers of polycaprolactone, poly(lactic-co-glycolic acid) and chitosan. As antimicrobials, the most frequently used have been antibiotics, though other antimicrobial substances, such as metallic ions, peptides and naturally derived products, have also been added to the nanopolymers. Polymeric nanomaterials containing antimicrobial compounds may be considered as a potential NGAS. Its relative efficacy, however, is not well understood since most of the existing evidence is derived from invitro or preclinical invivo studies.

SPECIES CHARACTERISTICS OF MICROBIAL ASSOCIATIONS IN THE COURSE OF TREATMENT OF 10 TO 14-YEAR-OLD CHILDREN WITH PLAQUE-INDUCED GINGIVITIS

Aim: Еvaluation of the species characteristics of subgingival biofilm microbial associations during treatment of children with plaque-induced gingivitis aged 10-14 years. Materials and methods: The subject of the study were 34 children with plaque-induced gingivitis. Three samples of subgingival biofilm are taken per patient: 1st sample - at the first visit; 2nd sample - two weeks later; 3rd the sample - one month later. Five teeth per child are selected from which a sample is taken for PCR testing for 9 control strains of subgingival microorganisms (A. actinomycetemcomitans, P. gingivalis, T. denticola, T. forsythia, P. intermedia, P. micros, F. nuleatum, E. nodatum, C. gingivalis). Results: C. gingivalis (green complex) is isolated in all examined children before the start of treatment and is indispensably observed in microbial associations with P. intermedia (orange complex). In other cases, associations with 7-8 microorganisms are observed and in a more complex composition. One month after the start of treatment, no periodontopathogens or only C. gingivallis as a single microorganism was is isolated in more than 55% of the samples examined. Microbial associations are predominantly 2 to 4 microorganisms.

Draft genome sequence of Limosilactobacillus fermentum isolated from subgingival biofilm.

Limosilactobacillus fermentum is generally considered beneficial for vaginal and digestive health. However, strains isolated from the oral cavity, especially from periodontitis lesions, have not been thoroughly studied. Here, we report the draft genome sequence of strain KHUD_007 isolated from the subgingival biofilm of a Korean patient with periodontitis.

Antimicrobial therapy using VIS plus water-filtered infrared-A as an alternative method to treat oral diseases.

Oral biofilm is the main cause of pathologies affecting the hard and soft oral tissues around teeth. Its main components are the periodontal pathogens and other bacteria of the supragingival and subgingival biofilm. Different alternative strategies that could be adjuvants to the usual periodontal treatments used to eliminate biofilms are available. One of these methods is antimicrobial photodynamic therapy using VIS and water-filtered infrared-A combined with a photosensitizer. In this review, different recent studies were collected to evaluate the antimicrobial effects of antimicrobial photodynamic therapy and the effectiveness of different types of photosensitizers.