Search

Objective To develop a reproducible subgingival microcosm biofilm model. Material and Methods Subgingival plaque samples were collected from four deep pockets ( probing pocket depth ≥6 mm) in each of seven patients with periodontitis and from shallow pockets ( probing pocket depth ≤3 mm) in two periodontally healthy donors. An active attachment model and a peptone medium (Thompson et. al., Appl Environ Microbiol 2015;81:8307-8314) supplemented with 30% serum was used. Biofilms were harvested at 2 and 4 weeks. DNA of dead cells was blocked for amplification by propidium monoazide treatment. Composition was analyzed using 16S rRNA gene amplicon pyrosequencing. Similarities between the biofilm samples were assessed by non-metric multidimensional scaling using the Bray-Curtis similarity index and similarity percentage analysis. Data from duplicate experiments, different biofilm sources and different biofilm age were compared. Results The non-metric multidimensional scaling revealed a strong clustering by the inoculum source, the donor and their periodontal status. Statistically significant differences were found between the sources of inoculum ( P=.0001) and biofilm age ( P=.0016). Furthermore, periodontitis biofilms (P) were distinct in composition from health-derived biofilms (H) by genera: Porphyromonas (P=19%; H=0%), Filifactor (P=10%; H=0%), Anaeroglobus (P=3%; H=0%), Phocaeicola (P=1.5%; H=0%), Parvimonas (P=19%; H=14%), Fusobacterium (P=2%; H=26%), Peptostreptococcus (P=20%; H=30%), Veillonella (P=7%; H=8%) and 57 other genera. Similarity distances ( Bray-Curtis) (mean 0.73, SD 0.15) and the Shannon diversity index (mean 2, SD 0.2) revealed no differences between duplicate experiments ( P=.121). Conclusion This biofilm model allows reproducible production of complex subgingival microbial communities. [ABSTRACT FROM AUTHOR]

The role of bacteria in causing apical periodontitis has been widely established, whilst the role of other microorganisms is studied less thoroughly. This systematic review and meta-analysis reviewed the literature for the prevalence and diversity of fungi in root canal infections. An extensive literature search was carried out in the Cochrane databases, EMBASE, MEDLINE, LILACS, Sci ELO and Web of Science. Additional studies were identified from six endodontic journals, four main endodontic textbooks and references of relevant papers. Selected clinical studies included sampling of necrotic pulps in permanent teeth and microbial analysis of these samples. Studies were critically appraised using the Joanna Briggs Institute Prevalence Critical Appraisal Checklist. Meta-analysis was performed using Meta XL. The screening of 1041 titles and abstracts and full-text reading yielded 54 studies. The overall prevalence of fungi in root canal infections was 7.5% ( CI 95%: 3.6-11.8%) in the inverse variance fixed effect heterogeneity model. Candida albicans was the most frequently isolated species. Significant heterogeneity was observed ( P < 0.001, I2 = 85.04%). Subgroup analyses based on geographical location, period of publication, type of infection, state of general health, communication with the oral cavity, type of sample and identification method revealed no factor influencing the prevalence. Better standardized techniques and a comprehensive analysis will reveal a more detailed and accurate representation of the prevalence and nature of fungi in root canal infections. [ABSTRACT FROM AUTHOR]

Aim To describe the application of a newly-developed in vitro model in which the diffusion of antimicrobials in oral biofilms can be studied. Methodology In a flow chamber consisting of three parallel feeding channels connected with each other by eight perpendicular side channels, multispecies biofilms were grown from saliva of a single donor for 48 h. The dimensions of the side channels were 100 μm × 100 μm × 5130 μm (H × W × L). When one or more side channels were filled with biofilm, the biofilms were stained with fluorescent stains. Then, one side-channel biofilm was selected and treated with phosphate buffered saline, 2% sodium hypochlorite (NaOCl), 17% ethylenediaminetetra-acetic acid ( EDTA) or modified salt solution ( MSS). Diffusion of the irrigants was observed by acquiring fluorescence images at 10× objective every 15 s for 30 min. Results It was possible to culture biofilms in the narrow (100 μm) channels. The biofilms varied in phenotype. In this model, no diffusion of Na OCl into the biofilms was seen after its application. Seventeen-percentage EDTA only diffused into the biofilm up to 200 μm in 30 min. MSS did diffuse in the biofilm over a distance of 450 μm within 2 min after a single application. Conclusions This new model enables the investigation of the diffusion of antimicrobials in biofilms. Other applications to improve our understanding of the characteristics of biofilms are now possible. [ABSTRACT FROM AUTHOR]

Aim To investigate the cytotoxicity of a modified salt solution ( MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. Methodology In a metabolic assay, fibroblasts derived from periodontal ligaments ( PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P < 0.05 was considered significant. Results The PDL fibroblasts remained metabolically active after challenges with MSS. Dentine powder did not alter the efficacy of MSS ( P > 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine ( CHX) or 2% sodium hypochlorite ( Na OCl) ( P > 0.05). Conclusions Modified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and Na OCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered. [ABSTRACT FROM AUTHOR]

Background and Objective To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. Methods Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases ( MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. Results Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin ( IL)-1β, IL-8, and monocyte chemotactic protein ( MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1β, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1β, MCP-1, and MMP-1 compared to periodontitis fibroblasts. Conclusions Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP -1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis. [ABSTRACT FROM AUTHOR]

In periodontitis, tissue damage results mainly from aberrant host responses to oral microorganisms. Fibroblasts can play an important role in this. Gingival fibroblasts do not develop tolerance against the lipopolysaccharide of Porphyromonas gingivalis, a keystone pathogen in periodontitis, which may partly explain the persistence of inflammation. However, besides lipopolysaccharide, live P. gingivalis possess numerous virulence traits to impair host-responses. We hypothesized that fibroblast-responsiveness to a bacterial challenge could be affected by live P. gingivalis. We investigated if inflammatory responses of gingival fibroblasts to P. gingivalis were altered, when the fibroblasts had encountered P. gingivalis previously. On consecutive days, primary human gingival fibroblasts were challenged twice for 6 h with live P. gingivalis, or fibroblasts were preincubated for 24 h with a lower concentration of live P. gingivalis and re-challenged for 6 h with a higher concentration. As the P. gingivalis capsule and proteases are involved in modulating host responses, we used encapsulated P. gingivalis W83 and a non-encapsulated mutant, and P. gingivalis ATCC33277 and a lys-gingipain and arg-gingipain mutant, to challenge fibroblasts. With all P. gingivalis-strains, interleukin-8 and monocyte chemoattractant protein-1 responses to the second challenge were less strong in fibroblasts that had been challenged with P. gingivalis before. These lower responses might correspond with higher interleukin-1 receptor agonist expression. Fibroblast responses to a second challenge were not influenced by 24 h preincubation. Reduced chemokine responses after consecutive potent P. gingivalis challenges indicate that gingival fibroblast responsiveness is affected by a previous bacterial encounter. In periodontitis, such reduced chemokine responses may impair chemotaxis and clearance of oral microorganisms, thereby leading to prolonged inflammatory responses and tissue damage. [ABSTRACT FROM AUTHOR]

Galassi F, Kaman WE, Anssari Moin D, van der Horst J, Wismeijer D, Crielaard W, Laine ML, Veerman ECI, Bikker FJ, Loos BG. Comparing culture, real-time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri-implant infections. J Periodont Res 2012; 47: 616-625. © 2012 John Wiley & Sons A/S Background and Objective: The aim of the study was to compare the detection of Porphyromonas gingivalis using a fluorescence resonance energy transfer (FRET) technology with commonly used diagnostic methods in salivary and subgingival plaque samples from subjects with dental implants. P. gingivalis was considered as a marker for a pathogenic microbiota. Material and Methods: Ninety-seven adult subjects were recruited, including periodontally healthy controls with no dental implants, implant controls with no peri-implant disease and patients with peri-implant disease. Saliva and subgingival/submucosal plaque samples were collected from all subjects and were analyzed using culture, real-time PCR and FRET technology employing P. gingivalis-specific substrates. Results: It was found that the P. gingivalis-specific substrates were highly suitable for detecting the presence of P. gingivalis in saliva and in subgingival plaque samples, showing comparable specificity to culture and real-time PCR. Conclusion: We applied the FRET technology to detect P. gingivalis in implant patients with or without an implant condition and in controls without implants. The technique seems suitable for detection of P. gingivalis in both plaque and saliva samples. However, with all three techniques, P. gingivalis was not very specific for peri-implantitis cases. Future work includes fine-tuning the FRET technology and also includes the development of a chair-side application. [ABSTRACT FROM AUTHOR]

Özok AR, Persoon IF, Huse SM, Keijser BJF, Wesselink PR, Crielaard W, Zaura E. Ecology of the microbiome of the infected root canal system: a comparison between apical and coronal root segments. International Endodontic Journal, 45, 530-541, 2012. Abstract Aim To evaluate the microbial ecology of the coronal and apical segments of infected root canal systems using a complete sampling technique and next-generation sequencing. Methodology The roots of 23 extracted teeth with apical periodontitis were sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5-V6 region. Results The sequences were classified into 606 taxa (species or higher taxon), representing 24 bacterial phyla or candidate divisions and one archaeal phylum. Proteobacteria were more abundant in the apical samples ( P < 0.05), whilst Actinobacteria were in significantly higher proportions in the coronal samples. The apical samples harboured statistically significantly more taxa than the coronal samples ( P = 0.01) and showed a higher microbial diversity. Several taxa belonging to fastidious obligate anaerobes were significantly more abundant in the apical segments of the roots compared with their coronal counterparts. Conclusions Endodontic infections are more complex than reported previously. The apical part of the root canal system drives the selection of a more diverse and more anaerobic community than the coronal part. The presence of a distinct ecological niche in the apical region explains the difficulty of eradication of the infection and emphasizes the need for new treatment approaches to be developed. [ABSTRACT FROM AUTHOR]

Scheres N, Laine ML, Sipos PM, Bosch-Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis . J Periodont Res 2011; 46: 407-416. © 2011 John Wiley & Sons A/S [ABSTRACT FROM AUTHOR]

Background and Objective: Porphyromonas gingivalis is a gram-negative obligate anaerobe that is strongly associated with severe periodontitis. Previous reports showed an association of P. gingivalis capsular polysaccharide with virulence. The K1 capsular polysaccharide was found to be more immunostimulatory than the other serotypes. Our objective was to explore the genetic background of the capsule biosynthesis (K-antigen) locus in a representative group of K1 serotype strains. Material and Methods: We used restriction fragment length polymorphism, polymerase chain reaction (PCR) and DNA sequencing to study the capsular polysaccharide locus in P. gingivalis K1 strains. For serotyping by double immunodiffusion and PCR we used 32 strains of P. gingivalis, including strains of all six known K serotypes. Results: All tested K1 strains showed high conservation of the capsular polysaccharide locus, although a DNA re-arrangement was found in two strains. Based on this information a K1-specific PCR-based serotyping assay was designed. The specificity and sensitivity of this test were confirmed using non-K1 P. gingivalis serotypes. Conclusion: The capsular polysaccharide locus of P. gingivalis is conserved but may vary slightly among K1 strains. The new K1 serotyping assay presented here is much faster than double immunodiffusion and can detect K1 strains in a very selective and sensitive way. This method may therefore be clinically relevant in the detection of the virulent P. gingivalis K1 serotype. [ABSTRACT FROM AUTHOR]

Introduction: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. Methods: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. Results: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. Conclusion: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens. [ABSTRACT FROM AUTHOR]

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment--protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids In the assembly of the photo-synthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls. [ABSTRACT FROM AUTHOR]

A letter to the editor about the report "Methods of Diagnosis and Treatment in Endodontics," which was endorsed by the Swedish Council on Health Technology Assessment (SBU), is presented.

In general, saliva is used for microbiota analysis in longitudinal studies, and several collection methods are being used. Using a robust sample collection procedure is important, as it may influence salivary composition. This study explored the comparability of the microbiota of swabbed and spit saliva. Twenty-two females participated in this cross-sectional study. The bacterial composition of the three saliva samples (swab collected by the participant (SW-P), swab collected by the researcher (SW-R), and spit (SP) was assessed by 16S rRNA gene amplicon sequencing. The bacterial composition of the swabbed and the spit saliva was significantly different irrespective of the operator, and Shannon diversity was significantly higher in spit saliva than in SW-P and SW-R. The salivary microbiota of spit and swabbed adult saliva differs significantly. Research on microbial composition therefore requires collection of similar saliva sample types in all study participants., (© 2021 The Authors. European Journal of Oral Sciences published by John Wiley & Sons Ltd on behalf of Scandinavian Division of the International Association for Dental Research.)

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., (© 2021 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.)

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., (© 2021 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.)

Background and Objective: Metal-based dental restorations with a subgingival outline may enhance plaque accumulation and bacterial colonization. This study aimed to investigate whether metal-based restorations influence the composition of subgingival microbiome., Material and Methods: Per subject one site with a metal-based restoration and one contra-lateral site without a restoration were selected on basis of radiographic bone loss ≤2 mm, restoration outline at sulcus level/subgingivally, pocket depth ≤4 mm, and no root canal treatments. Subgingival samples were collected with sterile paper-points, and microbial profiles were obtained by 16S rRNA gene amplicon sequencing. Restorations were sampled with an Arkansas-stone and the metal composition was determined using energy-dispersive X-ray spectroscopy., Results: A total of 22 sites from 11 subjects were included. No significant differences for the clinical parameters were found between the restored and unrestored sites. The average age of the restorations was 14.9 ± 7.1 years. Firmicutes was the most prevalent phylum at the restored sites (32% vs 20% of the reads of the unrestored sites, P = 0.016), and Actinobacteria at the unrestored sites (33% vs 18% of the reads of the restored sites, P = 0.01). Overall, sequences clustered into 573 operational taxonomic units (OTUs). Species richness of the restored sites was significantly higher than species richness of the unrestored sites (117 ± 32 and 96 ± 20 OTUs, respectively, P = 0.013). No associations between the metal composition and bacterial profiles were found., Conclusion: This study shows that metal-based restorations may enhance colonization of Firmicutes and the neighboring pocket may harbor more diverse microbial communities., (© 2019 The Authors. Journal of Periodontal Research Published by John Wiley & Sons Ltd.)

This study evaluated the inhibitory effects of lactams on Streptococcus mutans, Enterococcus faecalis, and Candida glabrata multispecies biofilm formation. γ-Alkylidene-γ-lactams 1, 2, and 3 [solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Glass coverslips were conditioned with either the lactams or 3.5% DMSO (control) for 1 h, inoculated with microbial cultures, and incubated for 48 h. To assess the effect of the lactams on biofilm formation, the following parameters were determined: the biofilm biomass (by both crystal violet staining and protein determination); the amount of insoluble polysaccharides of the extracellular matrix; and the number of viable and total cells [by both colony-forming unit counting and quantitative real-time PCR (qPCR)]. Data were analysed using one-way anova and post-hoc Tukey tests. Lactams 1, 2, and 3 promoted a statistically significant reduction in the amount of biofilm biomass, but only lactam 3 resulted in a statistically significant reduction in the number of attached viable E. faecalis. Both total protein content and the amount of extracellular polysaccharides decreased significantly. The effects of γ-alkylidene-γ-lactams 1, 2, and 3 on the inhibition of multispecies biofilm formation were evident by their ability to reduce the amount of protein and extracellular polysaccharides., (© 2018 Eur J Oral Sci.)

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., (© 2015 Eur J Oral Sci.)

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-1β, 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., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2013 John Wiley & Sons A/S.)

Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48-h E. faecalis biofilms were evaluated in colony-forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)(2) ] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double-hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections., (© 2012 Eur J Oral Sci.)

In clinical periodontology it is common practice to sample subgingival plaque from periodontitis patients and to search for the presence of alleged periodontal pathogens using routine laboratory techniques such as culture, DNA-DNA hybridization or real-time PCR. Usually, special attention is given to the recognition of 'red complex' microorganisms and to Aggregatibacter actinomycetemcomitans. Recently, molecular open-ended techniques have been introduced which are distinct from the more 'classical' approaches in that they do not preselect for certain species. In this study, we investigated to what extent the outcome of these techniques has changed our insight into the composition of the subgingival microbiota and whether this has consequences on clinical decision making. The open-ended approaches showed that the composition of subgingival plaque is much more complex than previously thought. Next to the 'classical' putative periodontal pathogens, several non-culturable and fastidious species are now recognized as being associated with periodontitis, thus enlarging the group of suspected periodontal pathogens. We conclude that routine analyses of subgingival plaque in the clinic are not necessarily of benefit to the patient., (© 2011 Eur J Oral Sci.)

In the oral cavity a balanced physiological response is essential for Streptococcus mutans to survive various types of external challenges. In this study we examined the role of the ClpP serine protease in the response of S. mutans towards sodium fluoride, sodium chloride, hydrogen peroxide, and chlorhexidine. By constructing a clpP promoter-green fluorescent protein reporter strain, we showed increased fluorescence intensities under all types of stress, indicating a need for ClpP under all these challenges. We constructed a clpP knockout mutant, which proved to be more sensitive to all the challenges than the wild-type strain. This knockout strain also displayed a reduced growth rate, hyperaggregation, and increased biofilm formation. Furthermore, an increased resistance to toxic levels of hydrogen peroxide and chlorhexidine after pre-incubation with sublethal levels of the corresponding compounds was found in the wild-type strain but not in the knockout mutant. In conclusion, ClpP is involved in the general stress response of S. mutans and assists the bacteria to resist killing through adaptation.

A structural study is described of the photoactive yellow protein (PYP) reconstituted with the chromophore derivative 3,4-dihydroxycinnamic acid. The crystal structure of PYP reconstituted with this chromophore at 1.16 A resolution is reported in space group P6(5). This is the first high-resolution structure of a photoreceptor containing a modified chromophore. The introduction of an extra hydroxyl group in the native chromophore (i.e. p-coumaric acid) appears to perturb the structure of the hybrid yellow protein only slightly. The chromophore is bound by the protein in two different conformations, separated by a rotation of 180 degrees of the catechol ring. In combination with available spectroscopic data, it is concluded that the caffeic acid chromophore binds to the protein in a strained conformation, which leads to a faster ejection from the chromophore-binding pocket upon pB formation.