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Background and Objective Little is known about the initiation of dysbiosis in oral biofilms, a topic of prime importance for understanding the etiology of, and preventing, periodontitis. The aim of this study was to evaluate the effect of different concentrations of crevicular and salivary peroxidase and catalase on dysbiosis in multispecies biofilms in vitro. Material and Methods The spotting technique was used to identify the effect of different concentrations of myeloperoxidase, lactoperoxidase, erythrocyte catalase, and horseradish peroxidase in salivary and crevicular fluid on the inhibitory effect of commensals on pathobiont growth. Vitality–quantitative real-time PCR was performed to quantify the dysbiotic effect of the peroxidases (adjusted to concentrations found in periodontal health, gingivitis, and periodontitis) on multispecies microbial communities. Results Agar plate and multispecies ecology experiments showed that production of hydrogen peroxide (H2O2) by commensal bacteria decreases pathobiont growth and colonization. Peroxidases at concentrations found in crevicular fluid and saliva neutralized this inhibitory effect. In multispecies communities, myeloperoxidase, at the crevicular fluid concentrations found in periodontitis, resulted in a 1-3 Log increase in pathobionts when compared with the crevicular fluid concentrations found in periodontal health. The effect of salivary lactoperoxidase and salivary myeloperoxidase concentrations was, in general, similar to the effect of crevicular myeloperoxidase concentrations. Conclusions Commensal species suppress pathobionts by producing H2O2. Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to dysbiosis by allowing the outgrowth of pathobionts.

Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that H2O2 production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.

The oral use of antimicrobial agents embedded in toothpastes and mouth rinses results in an oral microbial massacre with high amounts of dead bacteria in close proximity to few surviving bacteria. It was hypothesized that this provides the surviving pathogenic bacteria a large amount of dead microbial biomass as a nutritional source for growth (necrotrophy). This study demonstrated the necrotrophic growth of periodontal pathogens in the presence of different dead oral species. In addition, the presence of dead bacteria resulted in an outgrowth of several periodontal pathogens in complex multi-species biofilms. Additionally, upon contact with dead oral bacteria, virulence genes of P. intermedia and P. gingivalis were up-regulated (necrovirulence). This resulted in a more pronounced epithelial cytotoxicity (necrotoxicity). These findings indicate that presence of dead bacteria induce necrotrophy, necrovirulence and necrotoxicity in several oral pathogens.

Made available in DSpace on 2019-10-06T17:14:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 The aim of this study was to investigate the cytotoxic activity and inhibitory effect of terpinen-4-ol (T4ol) and carvacrol against single- and multi-species biofilms. The toxicity of each compound was tested on oral keratinocytes and evaluated by XTT assay. Inhibition and eradication of single-species biofilms were analyzed by crystal violet assay and the effect on multi-species biofilm composition was evaluated by qPCR. T4ol and carvacrol did not affect the epithelial cell viability, in contrast to chlorhexidine, which showed a high cytotoxic effect. Inhibition and eradication of single-species biofilms treated with T4ol and carvacrol were observed. The same inhibitory effect was observed for multi-species biofilms, especially on periodontal pathogens. In conclusion, specific concentrations of T4ol and carvacrol without toxicity towards the epithelial cells reduced the numbers of periodontal pathogens in single- and multi-species biofilms. Department of Oral Diagnosis and Surgery School of Dentistry São Paulo State University (Unesp) Department of Oral Health Sciences University of Leuven & Dentistry University Hospitals Leuven Department of Physiology and Pathology School of Dentistry São Paulo State University (Unesp) Department of Oral Diagnosis and Surgery School of Dentistry São Paulo State University (Unesp) Department of Physiology and Pathology School of Dentistry São Paulo State University (Unesp)

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

Objectives The objectives of this study are to identify oral commensal species which can inhibit the growth of the main periodontopathogens, to determine the antimicrobial substances involved in these inhibitory activities and to evaluate the influence of environmental factors on the magnitude of these inhibitions. Methods The spotting technique was used to quantify the capacity of 13 commensal species to inhibit the growth of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. By altering experimental conditions (distance between spots and size of spots and concentration of commensal and pathogen) as well as environmental factors (inoculation sequence, oxygen and nutrition availability) the influence of these factors was evaluated. Additionally, the mechanism of inhibition was elucidated by performing inhibition experiments in the presence of peroxidase, trypsin and pepsin and by evaluating acid production. Results Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus parasanguinis, Streptococcus mitis and Streptococcus oralis significantly inhibit the growth of all pathogens. The volume of the spots and concentration of the commensal have a significant positive correlation with the amount of inhibition whereas distance between the spots and concentration of the pathogen reduced the amount of inhibition. Inhibition is only observed when the commensal species are inoculated 24 h before the pathogen and is more pronounced under aerobic conditions. Hydrogen peroxide production by the commensal is the main mechanism of inhibition. Conclusion Bacterial antagonism is species specific and depending on experimental as well as environmental conditions. Blocking hydrogen peroxide production neutralizes the inhibitory effect. Clinical significance Identifying beneficial oral bacteria and understanding how they inhibit pathogens might help to unravel the mechanisms behind dysbiotic oral diseases. In this context, this study points towards an important role for hydrogen peroxide. The latter might lead in the future to novel preventive strategies for oral health based on improving the antimicrobial properties of commensal oral bacteria.

PubMedID: 26427036 Aim To evaluate the adjunctive effects of a Streptococcus oralis KJ3, Streptococcus uberis KJ2 and Streptococcus rattus JH145 containing probiotic tablet after scaling and root planing (SRP). Materials and Methods Forty-eight periodontitis patients were included in this double-blind, placebo-controlled clinical trial. After root planing, patients used either a placebo or a probiotic tablet twice a day for 12 weeks. The pocket probing depth (primary outcome measure), bleeding on probing and relative attachment levels were measured at baseline, 12 and 24 weeks. At baseline, 4, 8, 12 and 24 weeks, microbiological sampling was performed and plaque and gingival indices were recorded. Results The primary and secondary outcome measures were significantly (p < 0.05) improved at the 12- and the 24-week evaluation in both groups. However, no significant inter-group differences could be detected at any time point, except from the % of sites with plaque that were significantly lower in the probiotic group than in the control group at the 24-week evaluation. In addition, at the 12-week time point, the salivary Prevotella intermedia counts were significantly lower in the probiotic group. Conclusions No differences were detected when comparing the adjunctive use of a placebo or the investigated streptococci containing probiotic tablet after SRP. ClinicalTrials.gov Identifier: NCT02403960. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Subgingival debridement is the part of nonsurgical therapy which aims to remove the biofilm without intentionally removing the cementum or subgingival calculus. The objective of this review was to describe the end point of this therapy, the different methods used and how often it should be carried out. The literature shows that several methods are currently available for subgingival debridement, namely hand instrumentation, (ultra)sonic instrumentation, laser, photodynamic therapy and air-polishing. None of these methods seems superior to any other regarding clinical benefits or microbiological differences. However, less treatment discomfort is reported using laser, photodynamic therapy or air-polishing compared with hand- and/or (ultra)sonic instrumentation. Subgingival debridement can be carried out when, during supportive periodontal therapy, pockets of 5 mm or deeper are detected. ispartof: Periodontology 2000 vol:75 issue:1 pages:189-204 ispartof: location:Denmark status: published

Background: Only recently the concept of prebiotics has been introduced in oral health. Few potential oral prebiotics have already been identified in dual species competition assays, showing a stimulatory effect on beneficial bacteria and by this suppressing the outgrowth of pathogenic species. This study aimed to validate the effect of previously identified potential prebiotic substrates on multispecies cultures by shifting the biofilm composition towards a more beneficial species dominated microbiota. Methods: A chemostat culture containing 14 model oral bacterial species was used to grow biofilms for 24 hours which subsequently were treated with prebiotic solutions three times a day for 3 consecutive days. Further the influence of environmental factors such as pH, nutrient availability, oxygen concentration and prebiotic dose on the efficacy of the prebiotic substances was investigated. Results: Three potential prebiotic substrates N-acetyl-D-mannosamine, succinic acid and Met-Pro were able to bring the beneficial proportion to > 95%. While the pH of the prebiotic solution did not have an influence on the prebiotic effect, the interplay of nutrient availability, oxygen concentration and prebiotic treatment resulted in significant changes of the microbial composition identifying N-acetyl-D-mannosamine as the most promising oral prebiotic substrate. Showing a clear dose dependent effect, concentrations of N-acetyl-D-mannosamine of 1.0 and 1.5 M resulted in a biofilm composition of 97% beneficial species. Conclusion: Introducing the prebiotic concept in oral health might reveal a valid approach for treatment and prevention of oral diseases and promote oral health.

Aim To identify potential oral prebiotics that selectively stimulate commensal, albeit beneficial bacteria of the resident oral microbial community while suppressing the growth of pathogenic bacteria. Material and Methods Using Phenotype MicroArrays as a high-throughput method, the change in respiratory activity of 16 oral bacteria in response to 742 nutritional compounds was screened. Most promising prebiotic compounds were selected and applied in single species growth and biofilm formation assays, as well as dual species (beneficial-pathogen) competition assays. Results Increased respiratory activity could not always be related to an increase in growth or biofilm formation. Six compounds were used in dual species competition assays to directly monitor if selective nutritional stimulation of the beneficial bacterium results in the suppression of the pathogenic bacterium. Two compounds, beta-methyl-d-galactoside and N-acetyl-d-mannosamine, could be identified as potential oral prebiotic compounds, triggering selectively beneficial oral bacteria throughout the experiments and shifting dual species biofilm communities towards a beneficial dominating composition at in vitro level. Conclusion Our observations support the hypothesis that nutritional stimulation of beneficial bacteria by prebiotics could be used to restore the microbial balance in the oral cavity and by this promote oral health.

Dysbiosis in the periodontal microbiota is associated with the development of periodontal diseases. Little is known about the initiation of dysbiosis. It was hypothesized that some commensal bacteria suppress the outgrowth of pathobionts by H2O2 production. However, serum and blood components released due to inflammation can neutralize this suppressive effect, leading to the initiation of dysbiosis. Agar plate, dual-species and multi-species ecology experiments showed that H2O2 production by commensal bacteria decreases pathobiont growth and colonization. Peroxidase and blood components neutralize this inhibitory effect primarily by an exogenous peroxidase activity without stimulating growth and biofilm formation of pathobionts directly. In multi-species environments, neutralization of H2O2 resulted in 2 to 3 log increases in pathobionts, a hallmark for dysbiosis. Our data show that in oral biofilms, commensal species suppress the amounts of pathobionts by H2O2 production. Inflammation can neutralize this effect and thereby initiates dysbiosis by allowing the outgrowth of pathobionts.