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Peri-implant diseases are caused by bacterial biofilm colonizing implant surfaces. Prevention and management of peri-implant mucositis and peri-implantitis rely on effective biofilm removal. This study aimed to evaluate biofilm removal and cytocompatibility following chemo-mechanical surface decontamination of biofilm-coated titanium discs. Biofilm-coated (Streptococcus gordonii) discs, with either non-modified (smooth) or modified (rough) surfaces, were instrumented using a sterile gauze soaked in one out of four solutions: saline (NaCl), alkaline electrized water (AEW), citric acid (CA) or N-acetyl-l-cysteine (NAC). Non-contaminated, untreated titanium discs served as controls (C). Residual deposits (bacteria and gauze fibers) and cytocompatibility for osteoblast-like cells were evaluated using SEM and immunofluorescence. Cytotoxicity was assessed using WST-8 assay and immunofluorescence. All protocols were equally effective in removing bacteria from smooth surfaces, while AEW and CA were found to be superior at rough surfaces. AEW and NAC were superior in promoting cytocompatibility over NaCl. NAC and CA had a strong cytotoxic effect on osteoblast-like and fibroblast cells. In conclusion, AEW may be beneficial in the decontamination of implant surfaces, effectively removing bacterial biofilm and restoring cytocompatibility.

Objectives To analyse the long-term effect of plaque formation on implant abutments with an antibacterial coating and the ensuing host response in peri-implant tissues. Materials and methods Four implants were installed in each mandibular premolar region following tooth extraction in six dogs. Three months later, two test abutments with a titanium-bismuth-gallium (Ti-Bi-Ga) coating and two control titanium abutments were connected to the implants on each side of the mandible. After 2 months, ligatures were placed around the implants in one side of the mandible and plaque formation was allowed until the end of the experiment. The ligatures were removed after 4 weeks. Radiographs and microbiological samples were obtained from each implant site during the plaque formation period. Biopsies were obtained 8 months after abutment connection and prepared for histological analysis. Results The analysis did not reveal any statistically significant differences in bone loss, bacterial growth and size of inflammatory lesions between implant units with and without the Ti-Bi-Ga coating. Implant sites exposed to the short period of ligature-induced breakdown demonstrated more pronounced bone loss and bacterial growth than non-ligature sites. Conclusions It is suggested that a Ti-Bi-Ga coating does not prevent biofilm formation on the implant device and does not influence the ensuing host response in the adjacent peri-implant mucosa.

AIM To test the hypothesis that in the daily routine of a specialist clinic in endodontics that irrigation during root canal preparation with 3.0% NaOCl will result in fewer postoperative samples with cultivable bacteria than irrigation with 0.5% buffered NaOCl but, at the same time, will not result in a higher frequency of postoperative pain nor swelling. METHODOLOGY Two hundred ninety-eight patients were enrolled in the study and were randomly assigned into two groups - 0.5% NaOCl and 3% NaOCl. All endodontic diagnoses were included. Root canal treatment was performed, and bacterial sampling was carried out prior to root filling. The patients were requested to complete a form regarding pain and swelling seven days postoperatively. Fisher's exact test, Mann-Whitney U-test, Mantel-Haenszel chi-squared and the chi-squared test with a significance level of P

Beneficial effects of probiotics are relevant to the various potential properties of individual strains, and they may also relate to the original sources of the probiotic strains. This study aimed to characterize the potential probiotic properties of the strains originating from various sources for probiotics use in swine feeding. A total of 9 potential probiotic strains, seven lactobacilli and 2 bacilli, were examined for antimicrobial production against swine pathogens, adhesion and anti-adhesion of potential probiotic strains to IPEC-J2 cells, aggregation ability, host defense peptide expression, and hemolytic assay. The results highlight that all strains derived from different sources could exhibit probiotic properties, although different abilities were observed. L. rhamnosus SD11 exhibited the highest inhibitory effect against all pathogens compared to other strains. Bacillus licheniformis KMP-9, B. subtilis KMP-N004, and L. fermentum SD7 gave the highest internalization and that related to high abilities of exclusion, competition, and displacement inhibition to pathogens. Such strains also gave a higher co-aggregation to all pathogens compared to other potential probiotic strains. L. rhamnosus GG, L. fermentum SD7, L. rhamnosus SD4, and B. subtilis KMP-N004 had significantly higher pBD-2 mRNA expression than other strains. None of potential probiotic strains showed hemolytic activity. In conclusion, the strains derived from either humans or animals possessed desirable probiotic properties including inhibition against porcine pathogens, adhesion capacity to porcine enterocytes, anti-adhesion pathogens to porcine enterocytes, and modulated innate immunity. Results indicate that these probiotic strains may be good candidates for use in swine feeding to reduce the risk of infection.

The reported prevalence of periodontitis in children and adolescents varies considerably between populations globally. This cross-sectional study compares clinical and microbiological findings on 83 Somali immigrants and 96 non-Somali children aged 10-17 years old living in Trollhättan, Sweden. The clinical examination included registration of bleeding on probing, plaque, and calculus on incisors and first molars. The distance between cemento-enamel junction and bone level was measured on bitewing radiographs. Pooled microbiological samples (1 μL) were taken from the mesial surface of 16, 11, 31, 36, and analyzed by culture and real-time polymerase chain reaction for seven periodontal associated bacterial species. The Somali participants had poorer oral hygiene and more bleeding, plaque, and calculus. Ten of the Somali but none of the non-Somali participants showed periodontal breakdown (radiographical bone loss 3 mm), corresponding to a prevalence of 12% (95% CI: 5.9, 21.0%). The presence of A. actinomycetemcomitans was almost exclusively associated with Somali participants. Further, the JP2 clone was found in five Somalis (including two periodontitis cases) confirming the association of this clone with African populations. The Somali group showed significantly higher frequencies and numbers of Porphyromonas gingivalis and Treponema denticola, implying a mature and adult type of subgingival microbiota.

The objective of this in vitro study was to evaluate surface cleanness and cytocompatibility following mechanical instrumentation of biofilm-contaminated titanium surfaces. Titanium discs (non-modified [Ti(s)] and shot-blasted surfaces [Ti(r)]) contaminated with Streptococcus gordonii were instrumented using four different techniques: (i) gauze soaked in saline (GS), (ii) ultra-sonic device (US), (iii) rotating nickel-titanium brush (TiB), or (iv) air-polishing device (AP). Non-contaminated, untreated titanium disks were used as controls (C). Residual deposits and cytocompatibility for osteoblast-like cells were evaluated using scanning electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction. While the number of residual bacteria on Ti(s) discs was close to 0 in all treatment groups, significantly higher mean numbers of residual bacteria were observed on Ti(r) discs for GS (152.7 ± 75.7) and TiB (33.5 ± 22.2) than for US (0) and AP (0). Instrumentation with US resulted in deposition of foreign material (mean area% of foreign material: 3.0 ± 3.6% and 10.8 ± 9.6% for Ti(s) and Ti(r) discs, respectively). AP was the most effective decontamination procedure in reducing bacteria without depositing residual foreign material on Ti(r) discs. TiB and AP were superior methods in restoring cytocompatibility, although no method of mechanical decontamination resulted in pristine levels of cytocompatibility.

© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.Objective: This study assessed the prevalence of MRSA, ESBL and VRE in students from four dental schools in Europe. Methods: The hand, tongue and nostrils of the students who treated patients (study group) and who did not treat patients (control group) were sampled. After incubation in TSB and subculturing in the presence of 4 µg/ml oxacillin, positive cultures were identified for Staphylococcus aureus by Mannitol salt agar and agglutination tests. The presence of MRSA was confirmed by specific PCR on the species and on the SSCmec genes. ESBL and VRE were isolated using specific CHROMagar and confirmed using antibiotic sensitivity tests. Results: Of the 879 students who participated in this study (454 students which treated patients, 425 controls) a total of 50 students (5.7%) tested positive for a multi-drug resistant bacterium (MDRB); 13 (1.5%) students tested positive for MRSA, 26 (3.0%) for ESBL and 12 (1.4%) for VRE. No statistically significant differences were found between the students who treated patients compared to the control group for any of the MDRB and study centres, excluding MRSA carriage in the Italian student population. The use of antibiotics the year before sampling, was positively associated with the presence of an MDRB (OR 2.0; 95% Confidence Interval 1.10–3.68; p = 0.02). Conclusion: The risk for MDRB carriage and sequential transmission of MDRB for dental health care students and their patients were acceptably low.

Biofilm formation has become a significant problem in dental unit water lines (DUWLs). The formation of biofilms and microbial growth in DUWLs leads to an unacceptably high number of microorganisms in the water used for spraying, cooling, and ultrasonication procedures. These procedures form aerosols which can be inhaled by the patients, and consequently dentistry constitutes an area of specific concern for patient safety. In particular, older and immunocompromised patients are at risk of serious respiratory tract infections if the water contains pathogens such as Legionella pneumophila and Pseudomonas spp. In the EU it is recommended that the water in DUWLs should not exceed 200 colony-forming units (CFU) of heterotrophic bacteria (bacteria living on organic material) per milliliter of water to be acceptable in dental work. A number of efficient products are available on the market that can be applied onto dental units. New dental units are nowadays equipped with "inbuilt" systems. Such measures have resulted in an acceptable standard of water in 95% of the 1,200 dental units in the Public Dental Health Service of the Vastra Gotalands region of Sweden that were followed yearly for 4 years. For the majority of the remaining DUWLs with an unacceptable standard this is due to neglect or inappropriate routines for water-cleaning procedures. It is the ability to follow instructions rather than the cleaning procedure itself that is decisive if clinics and dental units are to have an appropriate standard of water in their systems.

Background This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1β secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay. Results Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1β secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1β secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1β, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed. Conclusions Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.

Background Epidemiological data from countries worldwide show a consistent pattern implying that a fraction of around 10% of those over 40–50 years in all populations will exhibit severe periodontitis with the potential risk of losing teeth during their life-time. The subgingival microbiota shows striking similarities between populations irrespective of disease severity and can only marginally explain the clinical pattern. It is also difficult to explain this pattern by genetic and acquired risk factors such as systemic disease (e.g. diabetes) or habits (e.g. smoking) even if they may have a confounding effect on the disease. Main text Inflammation of the gingiva appears to be a normal and physiological response to the presence of commensal bacteria along the gingival crevice and in the dental biofilm. Over many years of exposure to the dental biofilm, the chronic inflammation in the gingiva gradually results in a loss of attachment and bone loss. Numerous laboratory and clinical studies have provided insight into the potential role of determinants that are associated with periodontitis. However, it has been difficult to relate the findings to the pattern of the distribution of the disease observed in epidemiological studies. We propose a simple and parsimonious model that considers all the multitude of potential determinants as creating effectively random noise within the dental biofilm to which the tissues react by accumulating the effects of this noise. Conclusions We suggest that such a model can explain many of the epidemiological features of periodontal breakdown over time, and we discuss its clinical implications.

Background Prescribing in dental practice has a relatively small but important contribution to the quantity of antibiotics prescribed in primary care. This study aimed to analyse antibiotic prescribing in dentistry over time (2010–2016) in 4 different Northern European countries and their relative contribution to national outpatients consumption. Methods This retrospective study evaluated the frequency and number of national antibiotic prescriptions written by dentists in England, Scotland, Norway and Sweden. The consumption of such antibiotics was measured using WHO defined daily doses (DDDs), DDDs per 100,000 inhabitants per day (DIDs100,000). Results A total of more than 27 million prescriptions (27,026,599) archived between 2010 and 2016 from the four countries were analysed. The national contribution of Norwegian dentists to the total primary care prescription during this period was 8%. The corresponding figures for Sweden, Scotland and England were 7, 6, and 8%. Dental contribution to National antibiotic use in all four countries has decreased over the study time period for commonly prescribed antibiotics in dentistry, i.e., the beta-lactams (Phenoxymethyl penicillin/Amoxicillin) and metronidazole. There were less numbers of prescriptions by dentists in Norway and Sweden compared to England and Scotland. Marked differences in some classes of antibiotics were noted with Phenoxymethyl penicillin dominating in Sweden/Norway compared to Amoxicillin and Metronidazole in England/Scotland. In England and Scotland, dentists were the largest prescribers of metronidazole in primary care. Clindamycin prescriptions was higher in Norway and Sweden. Conclusion Noticeable differences exist in prescribing patterns for the management of oral infections. High levels of metronidazole use in England and Scotland also require further analysis. All countries over the study period showed a decrease in total numbers of antibiotics prescribed.

The aim was to investigate if hydrogen sulfide (H2S) induces the formation of the NLRP3 inflammasome and subsequent IL‐1β and IL‐18 secretion in human peripheral blood mononuclear cells (PBMCs) and in the human monocyte cell line THP1. Bacterial production of H2S has been suggested to participate in the inflammatory host response in periodontitis pathogenesis. H2S is a toxic gas with pro‐inflammatory properties. It is produced by bacterial degradation of sulfur‐containing amino acids, for example, cysteine. We hypothesize that H2S affects the inflammatory host response by inducing formation of the NLRP3 inflammasome and thereby causes the secretion of IL‐1ß and IL‐18. PBMCs from eight healthy blood donors, the human monocyte cell line THP1 Null, and two variants of the THP1 cell line unable to form the NLRP3 inflammasome were cultured in the presence or absence of 1 mM sodium hydrosulfide (NaHS) in 24‐well plates at 37°C for 24 hr. Supernatants were collected and the IL‐1β and IL‐18 concentrations were measured with DuoSet ELISA Development kit. PBMCs exposed to NaHS produced more IL‐1ß and IL‐18 than unexposed control cells (p = .023 and p = .008, respectively). An increase of extracellular potassium ions (K+) inhibited the secretion of IL‐1ß and IL‐18 (p = .008). Further, NaHS triggered the secretion of IL‐1ß and IL‐18 in human THP1‐Null monocytes (p = .0006 and p = .002, respectively), while the NaHS‐dependent secretion was reduced in the monocyte cell lines unable to form the NLRP3 inflammasome. Hence, the results suggest that NaHS induces the formation of the NLRP3 inflammasome and thus the secretion of IL‐1ß and IL‐18. Enhanced NLRP3 inflammasome‐dependent secretion of IL‐1β and IL‐18 in human mononuclear leukocytes exposed to NaHS in vitro is reported. This may be a mode for H2S to contribute to the inflammatory host response and pathogenesis of periodontal disease.

The objective of this study was to assess antibiotic susceptibility among predominant Gram-negative anaerobic bacteria isolated from periodontitis patients who 5 years prior had been subject to mechanical therapy with or without adjunctive metronidazole. One pooled sample was taken from the 5 deepest sites of each of 161 patients that completed the 5 year follow-up after therapy. The samples were analyzed by culture. A total number of 85 anaerobic strains were isolated from the predominant subgingival flora of 65/161 patient samples, identified, and tested for antibiotic susceptibility by MIC determination. E-tests against metronidazole, penicillin, amoxicillin, amoxicillin + clavulanic acid and clindamycin were employed. The 73/85 strains were Gram-negative rods (21 Porphyromonas spp., 22 Prevotella/Bacteroides spp., 23 Fusobacterium/Filifactor spp., 3 Campylobacter spp. and 4 Tannerella forsythia). These were all isolated from the treated patients irrespective of therapy procedures (+/−metronidazole) 5 years prior. Three strains (Bifidobacterium spp., Propionibacterium propionicum, Parvimonas micra) showed MIC values for metronidazole over the European Committee on Antimicrobial Susceptibility Testing break point of >4 μg/mL. All Porphyromonas and Tannerella strains were highly susceptible. Metronidazole resistant Gram-negative strains were not found, while a few showed resistance against beta-lactam antibiotics. In this population of 161 patients who had been subject to mechanical periodontal therapy with or without adjunct metronidazole 5 years prior, no cultivable antibiotic resistant anaerobes were found in the predominant subgingival microbiota.

We aimed to identify response patterns to non-surgical periodontal therapy and to investigate whether the new classification system for periodontitis reflects response to treatment after 1 yr. At baseline, data on sociodemographic status, smoking, and diabetes were obtained from participants with periodontal disease. Clinical periodontal data and subgingival plaque were also collected. Participants underwent non-surgical periodontal therapy, and after 3 and 12 months, clinical data were reassessed. Factor analyses, group-based-trajectory modeling, and mixed-effects regression models were used for data analysis. Factor analysis of the baseline periodontal parameters revealed two different periodontitis dimensions: 'moderate' and 'severe'. Two response patterns for each of these periodontitis dimensions were identified. Periodontal therapy had a beneficial effect on both 'moderate' and 'severe' periodontitis; however, individuals with higher levels of disease at baseline experienced greater treatment effect. Regarding the new classification system, while the staging component distinguished different levels of 'moderate' and 'severe' periodontitis before and after treatment, the grading component did not. This study shows the beneficial effect of non-surgical periodontal therapy on both 'moderate' and 'severe' periodontitis. However, the benefit was limited among individuals with low levels of disease. The new classification system did not adequately reflect the periodontal response to therapy in this patient group.

Periodontitis is a chronic inflammation that develops due to a destructive tissue response to prolonged inflammation and a disturbed homeostasis (dysbiosis) in the interplay between the microorganisms of the dental biofilm and the host. The infectious nature of the microbes associated with periodontitis is unclear, as is the role of specific bacterial species and virulence factors that interfere with the host defense and tissue repair. This review highlights the impact of classical virulence factors, such as exotoxins, endotoxins, fimbriae and capsule, but also aims to emphasize the often-neglected cascade of metabolic products (e.g., those generated by anaerobic and proteolytic metabolism) that are produced by the bacterial phenotypes that survive and thrive in deep, inflamed periodontal pockets. This metabolic activity of the microbes aggravates the inflammatory response from a low-grade physiologic (homeostatic) inflammation (i.e., gingivitis) into more destructive or tissue remodeling processes in periodontitis. That bacteria associated with periodontitis are linked with a number of systemic diseases of importance in clinical medicine is highlighted and exemplified with rheumatoid arthritis, The unclear significance of a number of potential “virulence factors” that contribute to the pathogenicity of specific bacterial species in the complex biofilm−host interaction clinically is discussed in this review.

Background: The mechanisms involved in the interplay between the bacteria and the host cells in periodontitis are not fully understood. Aim: To investigate the effect of the bacterial metabolite H2S on the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 from periodontitis patients and healthy controls, and to evaluate the composition of the subgingival microbiota with its capacity to produce H2S. Methods: Subgingival bacterial samples from patients with periodontitis (N=32) and healthy controls (N=32) were investigated for H2S production and bacterial composition. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence/absence of 1mM H2S for 24h and cytokine concentrations were measured. Results: Subgingival plaque from periodontitis patients had more H2S producing bacteria and produced more H2S, than healthy controls. PBMCs exposed to H2S secreted significantly more IL-1ß and IL-18 (p

Aim The aim of this study was to prospectively follow furcation tunnelled molars over a 5-year period of supportive periodontal therapy (SPT) and to identify factors associated with tooth loss. Materials and methods A total of 32 patients with 42 furcation tunnelled molars (all class III prior tunnelling) were recruited upon commencing SPT following active periodontal therapy. Clinical registrations, bacterial samples and standardised radiographs were taken at baseline, year 1 (no radiographs), 2 and 5. Total viable counts, total streptococci, Streptococcus sanguinis and mutans streptococci (MS) were identified through culture, a panel of periodontal pathogens through the checkerboard technique. Results After 5 years, 29 molars (69%) were still in function. Of the lost molars, eight were upper and five lower molars. Recurrent periodontal disease and caries were reasons for tooth loss. A multilevel regression analysis showed that a smoking habit, bleeding on probing and the presence of MS in furcations were associated with an increased risk of tooth loss. Conclusions Furcation tunnelled molars can in most cases be kept over a period of 5 years of SPT. A smoking habit, baseline bleeding scores and the presence of MS in the furcation were risk indicators for loss.

This study evaluated the associations between clinical, microbiological, and antibody activity manifestations of periodontitis in 123 adult rural Chinese subjects with no dental intervention. All participants were registered for full-mouth clinical attachment level (CAL) and pocket probing depth (PD) measurements, and microbial samples were taken from four sites and analyzed for 18 different bacterial species using the 'checkerboard'. Serum from each individual was analyzed to determine the antibody activity against the same 18 species. Exploratory factor analysis disclosed two microbial factors - Factor 1, consisting of seven species associated with periodontal health ('early colonizers'); and Factor 2, consisting of eight species associated with periodontitis ('putative periodontopathogens') - which explained 87% of the variation among the microbial variables. Factor 2 was consistently associated with disease-severity measures, whereas the 'early colonizer' factor was not. The antibody response showed weak or no correlations with bacterial load or with disease severity. We conclude that the bacteria investigated are resident in the subgingival plaque; that their load and proportions in the pocket may be ecologically driven; and that the antibody response is based on bacterial carrier state rather than on disease. The different antibody-response pattern found between the individuals may suggest that each individual could be classified as a good or a weak immune responder.

Objective To investigate the distribution of Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes among isolates from Thai chronic periodontitis patients. Design Forty-four adult Thai periodontitis patients were assessed by a full mouth recording for CAL, PPD, and BOP. Seventy-nine strains of A. actinomycetemcomitans were isolated from deep pockets on selective TSBV agar and 17 strains were isolated from shallow pockets. The strains were serotyped using PCR and subtyped using DGGE. Results The prevalence of A. actinomycetemcomitans was 84.1%. Non-serotypeable A. actinomycetemcomitans strains occurred equally frequent as serotypeable (54.5%); serotype a 18.2%, serotype c 15.9%, serotype e 9.1%, and serotype f 11.4%. Serotype b and d were not detected. A JP2 like strain but serotyped as c was isolated from two patients, and another two strains showed an 886 bp insertion on the ltx promoter of their A. actinomycetemcomitans isolates. DGGE typing disclosed 16 different subtypes among the non-serotypeable strains. Two of them (NS1 and NS2) were more common (12.7 and 10.1%) among the strains than the other 14 subtypes (˂5.1%). Most patients showed only one subtype (32.4%) but 29.7% had 2 and 3 different subtypes while 8.1% revealed 4 subtypes in one and the same deep pocket. Conclusion This study showed a greater subtype diversity of A. actinomycetemcomitans predominated by non-serotypeable strains than previously reported in an adult Thai population. It was also revealed for the first time that isolates with a 530 bp deletion or 886 bp insertion of the ltx promoter were serotyped as serotype c.

A high proportion of non-serotypeable isolates of Aggregatibacter actinomycetemcomitans among Thai periodontitis cases has been previously reported. The aim of this study was to investigate the expression of leukotoxin and toxicity, cytolethal distending toxin (Cdts), and internalization and the killing effect on fibroblasts by A. actinomycetemcomitans subtypes from Thai chronic periodontitis cases. A total of 96 A. actinomycetemcomitans strains from 37 periodontitis cases, previously serotyped with PCR and subtyped with DGGE, were examined for the presence of the ltx gene and cdt genes (cdtBC), and tested for leukotoxin expression, leukotoxicity, internalization, and apoptosis of fibroblast cells. The ltx gene was present in all isolates, while 84.4% showed the cdtBC gene. Two strains with a JP2-like ltx gene with a deletion of 530 bp in the promoter region, serotyped as c, showed virulence of similar magnitude to the JP2 strain. Furthermore, a higher virulence was found in the two non-serotypeable DGGE subtypes, NS1 and NS2, compared with the serotypeable strains (serotype a-f, serotype b and d were absent). Generally, the virulence of strains obtained from deep periodontal pockets was higher than those isolated from shallow non-bleeding pockets. A. actinomycetemcomitans subtypes isolated from adult Thais with chronic periodontitis showed a highly variable virulence, leukotoxin expression, leukotoxicity, internalization and apoptosis of fibroblast, and are regulated both genetically and environmentally.

Introduction: We are concerned that the dental profession, worldwide, has lost its way. We are a group of senior scientists – researchers, academics and intellectuals – from various parts of the world, with over 250 years’ combined experience of working to improve the oral health of communities. The group is entirely independent of any institution, government body or corporate entity. We met for a few days in March 2017 under the hospitality of Professor Emeritus Alfonso Escobar at his home in the Andes of Colombia, known as La Cascada Mare, to share our concerns about the future of dental care and dental education (Professor Richard Watt was unable to attend the meeting in Colombia, but was able to share an outline of his paper and to provide inputs into this declaration). Each of us prepared a detailed paper of our experiences over the last half century, our assessment of the problems and suggestions for the way forward. Each paper was discussed in detail (the full texts can be accessed at https://lacascada.pressbooks.com). The following statement represents our analysis of the problem and provides some recommendations about what should be done.

Owing to its molecular stability in body fluids, soluble urokinase-type plasminogen activator receptor (suPAR) is used as a biomarker for the level of systemic inflammation. This study compares the suPAR levels in serum with those in the saliva of adolescents and evaluates their association with the periodontal conditions. Adolescents identified as screen positive (n = 87) or screen negative (n = 73) for periodontitis had saliva and serum samples taken, along with subgingival plaque samples. The concentrations of suPAR were determined in saliva and serum, and 18 microbial species and the immunoglobulin response to them was evaluated. Factor analyses were used to reduce the number of variables within each of the domains of clinical, microbiological, and immunological findings. The median salivary suPAR concentration was 13.18 [(interquartile range (IQR): 6.20-23.36] μg l-1 and was not associated with the serum suPAR levels (median 2.05; IQR: 1.62-2.46 μg l-1 ). Linear regression analysis showed that the log10 (salivary suPAR concentration) was statistically significantly positively associated with the clinical phenotype 'Periodontitis Extent' (β = 0.28; 95% CI: 0.16-0.39) along with 'Putative periodontopathogens' (β = 0.65; 95% CI: 0.51-0.79). The study represents the first determination of salivary suPAR concentration in a larger well-defined adolescent population. Our results suggest the potential for clinical use of suPAR in saliva as an inflammatory risk indicator/biomarker of periodontitis.

The healthy human esophagus is colonized by bacteria similar to that of the oral mucosa. However, little is known about the microbiome of the esophagus in esophagitis or the possible role of bacteria in the inflammatory response.To survey bacterial diversity and compare the microbiome of the esophagus in subjects with gastro-esophageal reflux disease (GERD) and eosinophilic esophagitis (EoE).Seventeen subjects diagnosed with GERD and 10 with EoE underwent endoscopic examination with brush sampling and biopsies from the oral cavity, upper and lower esophagus. The samples were cultivated on agar plates, and bacterial growth was identified to the genus or species level and semi-quantified.Significantly higher numbers of bacterial groups or species were found in specimens from the lower esophagus in subjects with EoE compared to subjects with GERD (median 4 (range 1-7) vs. 2 (range 0-6), p .0014). Sixteen vs. 14 different bacterial groups or species were found in subjects with GERD and EoE, respectively, mostly in sparse or very sparse amounts. Alfa-streptococci (viridans streptococci) were the most common bacteria in both groups. Streptococci were present in all of the EoE-subjects but only in approximately 75% in lower esophagus of the GERD-subjects, regardless of the sampling method.Subjects with GERD had significantly less bacterial diversity in both oral and esophageal samples than EoE-subjects. Whether this discrepancy might be explained by an effect on the protective mucosal biofilm by the acidic content of the reflux in subjects with GERD remains unclear.

We review the literature on the oral microbiome and the role of the microbiota in the development of dental caries and periodontitis. While most research has been focused on identifying one or more specific determinants of these diseases, the results have provided limited predictive value and have not been able to explain the variation in the distribution of these diseases observed in epidemiological or clinical studies. Drawing on existing knowledge about the nature of the oral microbiota, we suggest that a stochastic model based on the Weiner process provides simple and parsimonious explanations for the pathogenesis of both caries and periodontitis, making few assumptions, and providing explanations for phenomena that have hitherto proved difficult, or have required complex arguments, to explain. These diseases occur as the result of the dental hard tissues and periodontal tissues integrating the random “noise” caused by normal metabolic activities of commensal microorganisms in the dental biofilm. The processes that result in the progression and regression of caries and periodontitis may be considered as “natural,” rather than pathological, even if, when left unchecked over long periods of time, they can result in the development of pathologies. The likelihood of progression or regression can be influenced by other determinants, but these processes will nevertheless occur in the absence of such influences. The distributional characteristics of the model approximate the findings of epidemiological studies indicating that, for both caries and periodontitis, there will be few sites affected in the early period after the eruption of the permanent dentition, but in those older there is an almost linear relationship with increasing age; furthermore, the longer a site survives without being affected, the less likely that it will be affected. We discuss the clinical and public health importance of these findings.

Periodontitis is a polymicrobial anaerobe infection. Little is known about the dysbiotic microbiota and the role of bacterial metabolites in the disease process. It is suggested that the production of certain waste products in the proteolytic metabolism may work as markers for disease severity. Hydrogen sulfide (H2S) is a gas produced by degradation of proteins in the subgingival pocket. It is highly toxic and believed to have pro-inflammatory properties. We aimed to study H2S production from subgingival plaque samples in relation to disease severity in subjects with natural development of the disease, using a colorimetric method based on bismuth precipitation. In remote areas of northern Thailand, adults with poor oral hygiene habits and a natural development of periodontal disease were examined for their oral health status. H2S production was measured with the bismuth method and subgingival plaque samples were analyzed for the presence of 20 bacterial species with the checkerboard DNA-DNA hybridization technique. In total, 43 subjects were examined (age 40-60 years, mean PI 95 ± 6.6%). Fifty-six percent had moderate periodontal breakdown (CAL 3 7 mm) and 35% had severe periodontal breakdown (CAL 7 mm) on at least one site. Parvimonas micra, Filifactor alocis, Porphyromonas endodontalis and Fusobacterium nucleatum were frequently detected. H2S production could not be correlated to periodontal disease severity (PPD or CAL at sampled sites) or to a specific bacterial composition. Site 21 had statistically lower production of H2S (p = 0.02) compared to 16 and 46. Betel nut chewers had statistically significant lower H2S production (p = 0.01) than non-chewers. Rapid detection and estimation of subgingival H2S production capacity was easily and reliably tested by the colorimetric bismuth sulfide precipitation method. H2S may be a valuable clinical marker for degradation of proteins in the subgingival pocket.

Purpose: Amoxicillin is commonly used in oral surgery for antimicrobial prophylaxis against surgical-site infection and bacteremia because of its effect on oral streptococci. The aim of this study was to determine whether amoxicillin reaches the break-point concentrations in saliva and has any effect on the salivary microbiota, colonizing bacteria on mucosal membranes and on the gingival crevice after a single dose of amoxicillin. Material and Methods: Twenty subjects received 2 g of amoxicillin, per os. The facultative and strictly anaerobic microflora, as well as the streptococcal microflora specifically, were followed from baseline and after 1, 4, and 24 hours. Samples were taken for microbial analysis from saliva, the dorsum of the tongue, and the gingival crevice, and were inoculated and cultured. Plasma samples and saliva samples were analyzed for amoxicillin concentrations (free and protein bound) using liquid chromatography and mass-spectrometry. Results: Amoxicillin was detected in concentrations over the break-point (>2 mu g/mL) of amoxicillin in plasma after 1 and 4 hours but not after 24 hours. The dose had a significant effect on the streptococci in the gingival crevice. Conclusion: A single dose given as prophylaxis to prevent a surgical-site infection results in a significant reducing effect on the oral streptococcal microflora in the gingival crevice and may have an impact on bacteria spreading into tissues and the bacteremia of streptococci.

Earlier this year, a group of us met in Colombia to reflect on the future of dentistry worldwide. We wanted to understand why oral health of most populations across the world had not improved, except in a few cases, despite massive growth in the number of dentists and paradental cadres trained each year in many countries and despite the employment of increasingly sophisticated technologies in the treatment of dental caries and periodontal diseases. We were concerned, moreover, that in many cases, the situation has worsened. Why is it, for example, that among populations with relatively easy access to dental care, the mortality of the dentition is considerable, with some 25%–30% of adults aged over 65 years being edentulous? And why is it that among populations with little or no access to dental care, and who often have poor oral hygiene, most people retain most of their teeth for most of their lives. Is there something wrong with the way that our profession has been operating?

OBJECTIVES: We aimed to investigate the association between dental caries and periodontitis among adolescents participating in a case-control study of periodontitis. In addition, we compared 2 analytical approaches to estimate the association: conventional regression and structural equation modelling (SEM).METHODS: Along with periodontal recordings, data were obtained on caries, just as subgingival plaque samples were collected. Sociodemographic information was collected with a questionnaire. We used factor analyses to express the many correlated clinical periodontal dimensions in a smaller number of factors. The association between caries (counts of enamel and dentin lesions, or dentin lesions only) and periodontitis was tested using negative binomial regression with robust variance (conventional regression) and weighted least squares (SEM) estimation.RESULTS: Factor analysis revealed 2 different latent periodontal variables: "extent" and "severity" of periodontitis. Using conventional regression, the "extent" of periodontitis was positively associated with higher counts of dentin caries lesions, even after adjustments for maternal education and subgingival microbial composition (rate ratio 1.34; 95% CI 1.07-1.68). The "severity" of periodontitis was associated with lower counts of enamel and dentin caries lesions (rate ratio 0.85 95% CI 0.77-0.92). The SEM revealed a positive association between periodontitis "extent" and number of dentin caries lesions (coefficient 0.29; P < .0001). The "severity" of periodontitis was negatively associated with enamel and dentin caries (coefficient -0.44; P < .0001).CONCLUSIONS: We found an association between caries and periodontitis among adolescents. The "severity" of periodontitis was negatively associated with enamel/dentin caries, whereas the "extent" of periodontitis was positively associated with dentin caries irrespective of the analytical approach employed.

In this chapter, the role of different microbial virulence factors in relation to the pathogenesis of periodontal diseases is addressed. These factors are molecules produced by pathogens and contribute to their pathogenicity by promoting colonization and affecting host response. The importance of different virulence factors in the life of the oral biofilm and the interplay with the host’s response is exemplified here by two of the major, and most well studied, periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Both of these microbes have great genetic intraspecies diversity and express a number of different virulence factors, which have the capacity to cause imbalance in the host’s response. A. actinomycetemcomitans is the major pathogen in aggressive forms of periodontitis (Fig. 4.1) that affect young individuals, while P. gingivalis is frequently detected in periodontal pockets of individuals with the chronic forms of the disease (Fig. 4.2). However, the role of these two bacteria in periodontal breakdown is still not entirely clear.

To compare the salivary microbial profile in children with oral clefts to matched non-cleft controls in a cross-sectional study. The cleft group consisted of 80 children aged 5 years and 144 age-matched non-cleft children were recruited as a control group. Stimulated whole saliva samples were collected and analysed with checkerboard DNA–DNA hybridisation using 12 pre-determined bacterial probes. Background data on caries, oral hygiene, dietary habits and fluoride exposure were collected from clinical examinations and questionnaires. Children in the cleft group had a significantly higher prevalence of dental caries but only minor differences in the salivary microbial profile were found. In general, the cleft-children displayed a lower prevalence of common commensal species (e.g. Streptococcus, Fusobacterium) when compared with the non-cleft controls. The differences were statistically significant (p

Varying cytokine responses of human gingival epithelial cells (HGECs) by Aggregatibacter actinomycetemcomitans subtypes have been found. Most studies have used reference strains, whereas a few has evaluated the cytokine expression in response to clinical subtypes of this bacterial species. This study aimed to examine whether there was any difference in cytokine responses of HGECs stimulated with cell wall extract (CWE) from A. actinomycetemcomitans subtypes included clinical strains from Thai adult periodontitis, various serotypes and non-serotypeable strains, strains from deep or shallow pockets, and reference serotype strains. Totally 50 clinical strains and 7 reference strains of A. actinomycetemcomitans were analyzed for the expression of IL-1β, IL-6, IL-8, and TNF-α mRNAs in HGECs by real time-PCR, and the IL-8 concentrations in cell-free supernatant measured using ELISA. An in vitro effect of released IL-8 on neutrophil migration was examined using transwell chambers. Result showed that among four cytokines studied, IL-8 mRNA was highly up-regulated by both clinical and reference strains. Serotype f revealed the highest expression compared to other serotypes. The JP2-like leukotoxin promoter gene and non-serotypeable (NS1 and NS2) demonstrated lower IL-8 responses compared to serotypeable strains, and IL-8 responses upon stimulation with clinical strains from deep pockets were also significantly lower than those isolated from shallow pockets (P

Background Urease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo. Methods The RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 μl of RUT test solution in the well of a micro-plate to which a 1 μl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 μl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test. Results The strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher scores compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46). Conclusion The rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease activity in bacterial strains in vitro and as a chair-side method for testing urease activity in site-specific supragingival plaque samples ex vivo.

Objectives To study the combined effect of mechanical and chemical cleansing on a 4-day biofilm grown intra-orally on titanium discs with different surface characteristics. Material and methods Twenty subjects used a splint with two metal plates in the upper jaw. Each plate was placed in the premolar-molar region and carried four titanium discs with four different surface characteristics (OsseoSpeed(™), TiOblast(™), experimental and turned surface). After 4 days of biofilm growth, the discs were cleaned mechanically and chemically with saline or chlorhexidine. Following cleansing, microbial samples were obtained and analysed by culture. The titanium discs were processed for scanning electron microscope (SEM) analysis. The experiment was repeated 3 days later using delmopinol or a mixture of essential oils during cleansing. Results The combination of mechanical and chemical cleansing was ineffective in complete biofilm removal from all four titanium discs. The microbiological analysis did not reveal any statistically significant differences between surface types or between cleaning agents regarding logarithmic mean counts of CFU for specific bacteria, aerobes, anaerobes or the TVC. Aerobes were more numerous than anaerobes on all surface types. The SEM analysis disclosed that the remaining biofilm on moderately rough surfaces (OsseoSpeed(™), TiOblast(™) and experimental) was complex and firmly attached, while the biofilm on turned surface had a pattern of spread bacteria forming less clusters. Conclusions Cleansing may call for prolonged time of chemomechanical debridement and/or more effective disinfectants to suppress biofilms on dental implant surfaces.

The aim of this study was to evaluate the plaque pH level and ureolytic activity among children and adults of Karen Hill tribes.Thirty-four children aged 6-10 years and 46 adults aged 20-38 years were interviewed regarding oral hygiene practices, sucrose intake and betel chewing. Caries experience (DMFT and DT), calculus, bleeding on probing (BoP) and Plaque index (PlI) were registered. Ureolytic activity in supragingival plaque was tested at two interproximal sites (11/12 and 41/42) with the rapid urease test (RUT). Registration of plaque pH was performed at two interproximal sites (15/16 and 31/41) before, during and 30 min after rinsing with an urea solution (0.25%). Four interproximal plaque samples (one from each quadrant) per individual were collected to test the bacterial composition using the checkerboard technique.Children and adults had similarly low DMFT and DT values. Children had a higher baseline pH and a higher ureolytic activity in the maxilla (p0.05) compared with adults. A significant correlation (r (2) = 0.63) was found between baseline pH and urease activity in the mandibular anterior teeth. Caries-free individuals had a higher baseline pH compared with caries active individuals in the anterior mandibular region (p0.01). The microbiological composition was characterized by an anaerobic low acidiogenic microbiota.Dental plaque pH is related to the ureolytic activity, which explains the low acidogenic plaque microflora and the low caries levels in the Karen population.

AIM: To assess the progression of attachment loss (AL) during a 2-year period according to the presence of JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans in a Ghanaian adolescent population.METHODS: A total of 500 adolescents (mean age 13.2 years, SD ± 1.5) were enrolled in the study. After 2 years, 397 (79.4%) returned for a periodontal re-examination, including the measurement of AL. The carrier status of the JP2 and non-JP2 genotypes of A. actinomycetemcomitans was evaluated in a baseline examination 2 years earlier.RESULTS: Individuals who carried the JP2 genotype of A. actinomycetemcomitans had a significantly increased risk [relative risk (RR) = 7.3] of developing AL ≥ 3 mm. The mean AL at the follow-up and the mean 2-year progression of AL were significantly higher in the JP2 genotype-positive group (n = 38) compared with the group positive for the non-JP2 genotypes of A. actinomycetemcomitans (n = 169), and the group of A. actinomycetemcomitans-negative individuals (n = 190). The JP2 genotype was strongly associated with the progression of AL ≥ 3 mm (OR = 14.3). The non-JP2 genotypes of A. actinomycetemcomitans were also, however, less pronounced, associated with the progression of AL ≥ 3 mm (OR = 3.4).CONCLUSION: The JP2 genotype of A. actinomycetemcomitans is strongly associated with the progression of AL.

Endodontic infections are complex diseases associated with apical tissue inflammation that is determined by microbial, immunological, and environmental factors. During the past years, the integration of research tools, including molecular techniques for identification, sophisticated in vitro modeling, and human microbiome analysis, has provided additional insight in the understanding of endodontic infections. Recent studies suggest that the basis for infections associated to root canals of teeth is polymicrobial in nature and includes the emergence of microbial colonization in form of biofilms. Biofilms deep seated in areas that are difficult to reach by mechanical treatment will enhance microbial virulence, antibiotic resistance, colonization potential, and resistance. Furthermore, with the advent of the human oral microbiome project, insights on the differences among oral microfloras in different individuals appear to have an important role in progressing endodontic infections. This chapter discusses the current data regarding the role that microbial biofilms play in endodontic infections, as well as its place in the current knowledge of endodontic microbiology. The complex relations between the root canal microflora and the inflammatory response in apical periodontitis are also highlighted in this chapter, as well as their implications in regard to the diagnosis and clinical management of endodontic infections.

BACKGROUND: To compare the oral microflora in preschool children attending a fluoride varnish program with a reference group receiving a standard oral health program without fluoride varnish applications. A second aim was to relate the microbial composition to the caries prevalence.METHODS: Five hundred seven 3-year-old children were enrolled from a cohort of 3403 preschool children taking part in a community based oral health project. Two hundred sixty-three of them had attended caries-preventive program with semi-annual applications of a fluoride varnish since the age of 1 year (test group) while 237 had received standard preventive care (reference group). Oral samples were collected with a sterile swab and analysed with checkerboard DNA-DNA hybridization using 12 pre-determined bacterial probes. Caries and background data were collected from clinical examinations and questionnaires.RESULTS: Gram-positive streptococci (S. intermedius, S. salivarius, S. oralis) were most frequently detected and displayed the highest counts in both groups. There were no significant differences between the groups concerning prevalence of any of the selected bacterial strains except for S. oralis that occurred less frequently in the reference group. In children with caries, V. parvula were significantly more common (p CONCLUSIONS: A 2-year community program with semi-annual fluoride varnish applications did not seem to significantly influence the oral microflora in preschool children.TRIAL REGISTRATION: www.controlled-trials.com (ISRCTN35086887) 20131216 'retrospectively registered'.