The microbiome of dental and peri-implant subgingival plaque during peri-implant mucositis therapy: A randomized clinical trial.

AIM: To assess the microbial effects of mechanical debridement in conjunction with a mouthrinse on sites with peri-implant mucositis and gingivitis. MATERIALS AND METHODS: Eighty-nine patients with peri-implant mucositis were included in a double-blinded, randomized, placebo-controlled trial with mechanical debridement and 1-month use of either delmopinol, chlorhexidine (CHX), or a placebo mouthrinse. Submucosal and subgingival plaque samples of implants and teeth were collected at baseline and after 1 and 3 months, processed for 16S V4 rRNA gene amplicon sequencing, and analysed bioinformatically. RESULTS: The sites with peri-implant mucositis presented with a less diverse and less anaerobic microbiome. Exposure to delmopinol or CHX, but not to the placebo mouthrinse resulted in microbial changes after 1 month. The healthy sites around the teeth harboured a more diverse and more anaerobe-rich microbiome than the healthy sites around the implants. CONCLUSIONS: Peri-implant sites with mucositis harbour ecologically less complex and less anaerobic biofilms with lower biomass than patient-matched dental sites with gingivitis while eliciting an equal inflammatory response. Adjunctive antimicrobial therapy in addition to mechanical debridement does affect both dental and peri-implant biofilm composition in the short term, resulting in a less dysbiotic subgingival biofilm.

Reprocessing 16S rRNA Gene Amplicon Sequencing Studies: (Meta)Data Issues, Robustness, and Reproducibility.

High-throughput sequencing technology provides an efficient method for evaluating microbial ecology. Different bioinformatics pipelines can be used to convert 16S ribosomal RNA gene amplicon sequencing data into an operational taxonomic unit (OTU) table that is used to analyze microbial communities. It is important to assess the robustness of these pipelines, each with specific algorithms and/or parameters, and their influence on the outcome of statistical tests. Articles with publicly available datasets on the oral microbiome were searched for, and five datasets were retrieved. These were from studies on changes in microbiota related to smoking, oral cancer, caries, diabetes, or periodontitis. Next, the data was processed with four pipelines based on VSEARCH, USEARCH, mothur, and UNOISE3. OTU tables were rarefied, and differences in α-diversity and ß-diversity were tested for different groups in a dataset. Finally, these results were checked for consistency among these example pipelines. Of articles that deposited data, only 57% made all sequencing and metadata available. When processing the datasets, issues were encountered, caused by read characteristics and differences between tools and their defaults in combination with a lack of detail in the methodology of the articles. In general, the four mainstream pipelines provided similar results, but importantly, P-values sometimes differed between pipelines beyond the significance threshold. Our results indicated that for published articles, the description of bioinformatics methods and data deposition should be improved, and regarding reproducibility, that analysis of multiple subsamples is required when using rarefying as library-size normalization method.

Submucosal microbiome of peri-implant sites: A cross-sectional study.

AIM: To study the peri-implant submucosal microbiome in relation to implant disease status, dentition status, smoking habit, gender, implant location, implant system, time of functional loading, probing pocket depth (PPD), and presence of bleeding on probing. MATERIALS AND METHODS: Biofilm samples were collected from the deepest peri-implant site of 41 patients with paper points, and analysed using 16S rRNA gene pyrosequencing. RESULTS: We observed differences in microbial profiles by PPD, implant disease status, and dentition status. Microbiota in deep pockets included higher proportions of the genera Fusobacterium, Prevotella, and Anaeroglobus compared with shallow pockets that harboured more Rothia, Neisseria, Haemophilus, and Streptococcus. Peri-implantitis (PI) sites were dominated by Fusobacterium and Treponema compared with healthy implants and peri-implant mucositis, which were mostly colonized by Rothia and Streptococcus. Partially edentulous (PE) individuals presented more Fusobacterium, Prevotella, and Rothia, whereas fully edentulous individuals presented more Veillonella and Streptococcus. CONCLUSIONS: PPD, implant disease status, and dentition status may affect the submucosal ecology leading to variation in composition of the microbiome. Deep pockets, PI, and PE individuals were dominated by Gram-negative anaerobic taxa.

Manipulation of Saliva-Derived Microcosm Biofilms To Resemble Dysbiotic Subgingival Microbiota.

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and reestablish a health-associated microbiota, a high-throughput in vitro multispecies biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rRNA gene amplicon sequencing as well as measurement of dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principal-component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis to saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder a microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies.IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed targeting reversing dysbiosis and restoring host-compatible microbiota rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in a dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large-scale evaluation of strategies that can potentially modulate microbial ecology.

Oral microbiome-systemic link studies: perspectives on current limitations and future artificial intelligence-based approaches.

In the past decade, there has been a tremendous increase in studies on the link between oral microbiome and systemic diseases. However, variations in study design and confounding variables across studies often lead to inconsistent observations. In this narrative review, we have discussed the potential influence of study design and confounding variables on the current sequencing-based oral microbiome-systemic disease link studies. The current limitations of oral microbiome-systemic link studies on type 2 diabetes mellitus, rheumatoid arthritis, pregnancy, atherosclerosis, and pancreatic cancer are discussed in this review, followed by our perspective on how artificial intelligence (AI), particularly machine learning and deep learning approaches, can be employed for predicting systemic disease and host metadata from the oral microbiome. The application of AI for predicting systemic disease as well as host metadata requires the establishment of a global database repository with microbiome sequences and annotated host metadata. However, this task requires collective efforts from researchers working in the field of oral microbiome to establish more comprehensive datasets with appropriate host metadata. Development of AI-based models by incorporating consistent host metadata will allow prediction of systemic diseases with higher accuracies, bringing considerable clinical benefits.

Apical Periodontitis Is Associated with Elevated Concentrations of Inflammatory Mediators in Peripheral Blood: A Systematic Review and Meta-analysis.

INTRODUCTION: Apical periodontitis (AP), except for the local known consequences, may also be a systemic burden. Circulating inflammatory mediators that are released to sustain the AP lesion can in theory harm other bodily tissues. The aim of this systematic review was to summarize the existing evidence on the influence of AP on the peripheral blood levels of inflammatory mediators and markers of systemic stress. METHODS: A search of MEDLINE-PubMed, Embase, and Cochrane was conducted up to and including February 2019 to identify studies in 5 different languages. The Newcastle-Ottawa Scale was used for quality assessment of the included studies. RESULTS: Twelve of the 20 included studies were case-control studies, and 8 were intervention studies. The data of all the included studies were analyzed descriptively, whereas the data of 11 studies were available for meta-analyses. The study designs were heterogeneous. Nevertheless, the meta-analyses revealed statistically significant differences in C-reactive protein, interleukin 6, and asymmetric dimethylarginine levels between AP subjects and controls in peripheral blood. In addition, the concentration of C3 complement fragment in peripheral blood was significantly lower after the treatment and resolution of AP than before. CONCLUSIONS: The existing literature indicates that AP adds on to systemic inflammation by elevating C-reactive protein, interleukin 6, asymmetric dimethylarginine, and C3 levels. In order to overcome the issue of large variation between study designs, future studies should have clear inclusion criteria, preferably larger cohorts, adequate follow-up of all subjects, and a thorough presentation of the data to enable further exploration of the possible burden of AP on general human health. Nevertheless, there is now stronger evidence that AP contributes to low-grade systemic inflammation.

Resistance and resilience to experimental gingivitis: a systematic scoping review.

BACKGROUND: This systematic scoping review aimed to identify changes in biomarkers of microbiological, immunological and biochemical origin during experimental gingivitis (EG) studies that might indicate resistance and resilience. METHODS: The term 'experimental gingivitis' was run in PubMed from inception to April 11th, 2018. From the 411 studies retrieved, 22 studies were included for this review. RESULTS: Studies reporting data on biomarker changes during and after full mouth EG trial were included. Two studies reported findings on changes in biomarkers of microbiological, 12 on immunological and eight on biochemical origin. Changes were reported in the induction phase, and occasionally in the resolution phase. The microbiological composition of both supragingival and subgingival dental plaque changed over the course of EG to a more pathogenic direction, but showed a shift back to a more normal composition. This indicates resilience of the oral microbiome. For immunological biomarkers, it was challenging to retrieve a robust pattern of changes across multiple studies. IL-1ß and IL-6 in saliva and in gingival crevicular fluid increased during induction phase and returned in the resolution phase below baseline values. The biochemical parameters cystatin-SN, cystatin-S and lactoferrin in saliva were increased at the end of induction phase, however also here no clear pattern emerged based on all available studies. CONCLUSIONS: More research is needed to investigate which microbiological, immunological, and biochemical biomarkers can be useful for future investigations into the resistance and resilience of the oral cavity to experimental gingivitis.

Multi-species oral biofilm promotes reconstructed human gingiva epithelial barrier function.

Since the oral mucosa is continuously exposed to abundant microbes, one of its most important defense features is a highly proliferative, thick, stratified epithelium. The cellular mechanisms responsible for this are still unknown. The aim of this study was to determine whether multi-species oral biofilm contribute to the extensive stratification and primed antimicrobial defense in epithelium. Two in vitro models were used: 3D reconstructed human gingiva (RHG) and oral bacteria representative of multi-species commensal biofilm. The organotypic RHG consists of a reconstructed stratified gingiva epithelium on a gingiva fibroblast populated hydrogel (lamina propria). Biofilm was cultured from healthy human saliva, and consists of typical commensal genera Granulicatella and major oral microbiota genera Veillonella and Streptococcus. Biofilm was applied topically to RHG and host-microbiome interactions were studied over 7 days. Compared to unexposed RHG, biofilm exposed RHG showed increased epithelial thickness, more organized stratification and increased keratinocyte proliferation. Furthermore biofilm exposure increased production of RHG anti-microbial proteins Elafin, HBD2 and HBD3 but not HBD1, adrenomedullin or cathelicidin LL-37. Inflammatory and antimicrobial cytokine secretion (IL-6, CXCL8, CXCL1, CCL20) showed an immediate and sustained increase. In conclusion, exposure of RHG to commensal oral biofilm actively contributes to RHG epithelial barrier function.

Effects of bacterial physiological states and bacterial species on host-microbe interactions.

This study investigated how the physiological states of Aggregatibacter actinomycetemcomitans (Aa) and Streptococcus mitis affect their intracellular invasion capabilities and the resulting host cell responses. The physiological states included two forms of planktonic states, floating or sedimented (by centrifugation) and the biofilm state (with centrifugation). Confluent epithelial Ca9-22 cells were challenged with floating or sedimented planktonic cultures, or with 24-h biofilms for 3 h. The results show that intracellular invasion efficiencies were clearly affected by the bacterial physiological states. For both bacterial species, the sedimented-cells displayed 2-10 times higher invasion efficiency than the floating-cells (p < 0.05). The invasion efficiency of Aa biofilms was three fold lower than sedimented cells, whereas those of S. mitis biofilms were similar to sedimented cells. Unlike invasion, the metabolic activities of Ca9-22 were unaffected by different bacterial physiological states. However, Aa biofilms induced higher IL-1ß expression than planktonic cultures. In conclusion, different bacterial physiological states can affect the outcomes of (in vitro) host-microbe interaction in different ways.

Subgingival microbiome of rheumatoid arthritis patients in relation to their disease status and periodontal health.

OBJECTIVE: Rheumatoid arthritis (RA) and periodontitis are chronic inflammatory diseases that share common risk factors. However, the bidirectional relationship between RA and periodontal disease is not fully understood. This study was undertaken to describe the bacterial component of the subgingival microbiome in RA patients and to relate this to RA disease activity and periodontal status. METHODS: Patients with chronic established RA (N = 78) were periodontally examined and their subgingival plaque samples were collected; their clinical and laboratory data on RA status and medication were obtained from medical records. Bacterial DNA was quantified by universal 16S rDNA qPCR, and Porphyromonas gingivalis by species-specific qPCR. For microbiome assessment, 16S rDNA amplicon sequencing was performed. RESULTS: Active RA was diagnosed in 58% of the patients and periodontitis in 82% (mild: 9%, moderate: 55%, severe: 18%). P. gingivalis was present in 14% of the samples. Different levels of gingival bleeding, periodontal probing depth, RA disease status, prednisolone use and smoking were associated with significantly different microbiome compositions. Two subgingival microbial community types were discerned. CONCLUSION: In RA patients with active disease, anti-inflammatory medication as part of RA therapy was associated with better oral health status and a healthier subgingival microbiome compared to that of RA patients in remission, especially those in remission who were current smokers. RA patients in remission with current smoking status may particularly benefit from a systematic periodontal treatment program. The potential role of microbial community types in patient stratification and personalized therapy should be assessed in longitudinal studies.

Antimicrobial photodynamic therapy - what we know and what we don't.

Considering increasing number of pathogens resistant towards commonly used antibiotics as well as antiseptics, there is a pressing need for antimicrobial approaches that are capable of inactivating pathogens efficiently without the risk of inducing resistances. In this regard, an alternative approach is the antimicrobial photodynamic therapy (aPDT). The antimicrobial effect of aPDT is based on the principle that visible light activates a per se non-toxic molecule, the so-called photosensitizer (PS), resulting in generation of reactive oxygen species that kill bacteria unselectively via an oxidative burst. During the last 10-20 years, there has been extensive in vitro research on novel PS as well as light sources, which is now to be translated into clinics. In this review, we aim to provide an overview about the history of aPDT, its fundamental photochemical and photophysical mechanisms as well as photosensitizers and light sources that are currently applied for aPDT in vitro. Furthermore, the potential of resistances towards aPDT is extensively discussed and implications for proper comparison of in vitro studies regarding aPDT as well as for potential application fields in clinical practice are given. Overall, this review shall provide an outlook on future research directions needed for successful translation of promising in vitro results in aPDT towards clinical practice.

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures.

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.

Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces.

UNLABELLED: Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE: Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults.

A cross-sectional observational study was conducted to evaluate interindividual biochemical variation in unstimulated whole saliva in a population of 268 systemically healthy young students, 18-30 yr of age, with no apparent caries lesions or periodontal disease. Salivary flow rate, protein content, pH, buffering capacity, mucins MUC5B and MUC7, albumin, secretory IgA, cystatin S, lactoferrin, chitinase, amylase, lysozyme, and proteases were measured using ELISAs and enzymatic activity assays. Significant differences were found between male and female subjects. Salivary pH, buffering capacity, protein content, MUC5B, secretory IgA, and chitinase activity were all lower in female subjects compared with male subjects, whereas MUC7 and lysozyme activity were higher in female subjects. There was no significant difference between sexes in salivary flow rate, albumin, cystatin S, amylase, and protease activity. Principal component analysis (PCA) and spectral clustering (SC) were used to assess intervariable relationships within the data set and to identify subgroups. Spectral clustering identified two clusters of participants, which were subsequently described. This study provides a comprehensive overview of the distribution and inter-relations of a set of important salivary biochemical variables in a systemically healthy young adult population, free of apparent caries lesions and periodontal disease. It highlights significant gender differences in salivary biochemistry.

LuxS signaling in Porphyromonas gingivalis-host interactions.

Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1ß, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.

Novel tea polyphenol-modified calcium phosphate nanoparticle and its remineralization potential.

Tea polyphenols (TP) are not only potent antimicrobial and antioxidant agents but also effective modifiers in the formation of nanosized crystals. Since nano-hydroxyapatite (n-HA) is known to enhance remineralization of dental hard tissue, our aims were to synthesize nanosized calcium phosphate particles incorporating TP and to test their potential as caries preventive agent. An ammonia water diffusion method was used to synthesize nanosized calcium phosphate particles (TP-CaP) in the presence of various amounts of TP. The resultant products were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The remineralization potential of the nano TP-CaP was then investigated in a 12-day pH-cycling model. Nano TP-CaP slurries, at pH 7.0 and pH 5.5, were applied onto preformed enamel lesions 4 × 3 min per day. n-HA slurries at pH 7.0 and pH 5.5 were used as positive controls, and deionized water was served as a negative control. SEM showed nanosized particles were only formed at 27 mg/mL of TP. Further characterization of the nanosized particles revealed the components were amorphous calcium phosphate, HA, and TP. Both surface microhardness and transverse microradiography analyses showed that nano TP-CaP at pH 5.5, but not at pH 7.0, significantly enhanced remineralization, to the same extent as the n-HA controls. Furthermore, significantly higher amount of TP was found in the supernatant of TP-CaP at pH 5.5 than those at pH 7.0. Since TP can inhibit bacterial growth and enzyme activities, the novel nanosized TP-CaP particle, at low pH, is a potential dual-functional-remineralization and antibacteria-product.

Influence of titanium on in vitro fibroblast-Porphyromonas gingivalis interaction in peri-implantitis.

AIM: Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenesis of peri-implantitis remains unclear. We aimed to determine the in vitro inflammatory responses of peri-implant granulation tissue fibroblasts (PIGFs) to titanium particles alone and in the presence of viable Porphyromonas gingivalis. MATERIALS & METHODS: Peri-implant granulation tissue fibroblasts were challenged either with TiO2 particles, P. gingivalis or a combination of TiO2 particles and P. gingivalis. Gene expression and protein production of pro-inflammatory mediators by PIGFs were measured with PCR and ELISA, respectively. RESULTS: Higher doses of TiO2 were toxic to PIGFs and in sub-toxic doses, TiO2 caused an increase in gene expression of tumour necrosis factor (TNF)-A and increased protein production of TNF-α, interleukin (IL)-6 and IL-8. A challenge with P. gingivalis alone induced gene expression of TNF-A, IL-1ß, IL-6 and IL-8. A combined challenge with TiO2 and P. gingivalis caused a stronger increase in gene expression of TNF-A and protein production of TNF-α and MCP-1 than P. gingivalis alone. CONCLUSIONS: TiO2 particles and P. gingivalis, individually, can induce pro-inflammatory responses in PIGFs. Furthermore, TiO2 particles and viable P. gingivalis further enhance gene expression and production of TNF-α by PIGFs. Therefore, Ti wear particles in the peri-implant tissues in combination with P. gingivalis infection may contribute to the pathogenesis of peri-implantitis by enhancing the inflammation in peri-implant tissues.

Subgingival microbiome in smokers and non-smokers in periodontitis: an exploratory study using traditional targeted techniques and a next-generation sequencing.

AIM: To compare the results of two targeted techniques to an open-ended technique in periodontitis patients, differentiated on the basis of smoking habit. MATERIALS & METHODS: Thirty periodontitis patients (15 smokers and 15 non-smokers) provided subgingival plaque samples for 16S rRNA gene amplicon sequencing, culturing and quantitative polymerase chain reaction (qPCR). RESULTS: No differences were found in the composition of the subgingival microbiome between smokers and non-smokers with culture and qPCR. With pyrosequencing, operational taxonomic units (OTUs) classified to genera Fusobacterium, Prevotella and Selenomonas were more abundant in smokers, while OTUs belonging to the genera Peptococcus and Capnocytophaga were more abundant in non-smokers. Principal coordinate analysis identified two clusters; one was composed mainly of smokers (80%) and revealed significantly lower taxonomic diversity, higher attachment loss and higher proportion of the genera Fusobacterium, Paludibacter and Desulfobubus. CONCLUSION: In periodontitis, there is a difference in the composition of the subgingival microbiome between smokers and non-smokers, as revealed by pyrosequencing. This difference was not identified by the targeted techniques. Low taxonomic diversity was associated with higher disease severity, especially in smokers. This supports the hypothesis of the ecological microbial-host interaction in the severity of periodontal disease.

Application of an active attachment model as a high-throughput demineralization biofilm model.

OBJECTIVES: To investigate the potential of an active attachment biofilm model as a high-throughput demineralization biofilm model for the evaluation of caries-preventive agents. METHODS: Streptococcus mutans UA159 biofilms were grown on bovine dentine discs in a high-throughput active attachment model. Biofilms were first formed in a medium with high buffer capacity for 24h and then subjected to various photodynamic therapies (PACT) using the combination of Light Emitting Diodes (LEDs, Biotable(®)) and Photogem(®). Viability of the biofilms was evaluated by plate counts. To investigate treatment effects on dentine lesion formation, the treated biofilms were grown in a medium with low buffer capacity for an additional 24h. Integrated mineral loss (IML) and lesion depth (LD) were assessed by transversal microradiography. Calcium release in the biofilm medium was measured by atomic absorption spectroscopy. RESULTS: Compared to the water treated control group, significant reduction in viability of S. mutans biofilms was observed when the combination of LEDs and Photogem(®) was applied. LEDs or Photogem(®) only did not result in biofilm viability changes. Similar outcomes were also found for dentine lesion formation. Significant lower IML and LD values were only found in the group subjected to the combined treatment of LEDs and Photogem(®). There was a good correlation between the calcium release data and the IML or LD values. CONCLUSIONS: The high-throughput active attachment biofilm model is applicable for evaluating novel caries-preventive agents on both biofilm and demineralization inhibition. PACT had a killing effect on 24h S. mutans biofilms and could inhibit the demineralization process.

Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.