Antimicrobial capacity of Leucocyte-and Platelet Rich Fibrin against periodontal pathogens.

Various studies have described the biological properties of the Leucocyte- and Platelet Rich Fibrin (L-PRF) such as the antimicrobial effect against wound bacteria, but less is known about the effect against periodontal pathogens. The aim of this study was to evaluate the antibacterial properties of the L-PRF membrane and L-PRF exudate against the main periopathogens cultured on agar plates and in planktonic solution. This study demonstrated the antibacterial effect of the L-PRF membrane against P. intermedia, F. nucleatum, and A. actinomycetemcomitans, but especially against P. gingivalis. The L-PRF exudate also showed a strong inhibition against P. gingivalis on agar plates. No inhibition could be observed for the other bacterial strains. Moreover, L-PRF exudate decreased the number of viable P.gingivalis in a planktonic solution in a dose-dependent way. However, A. actinomycetemcomitans showed an increased growth in planktonic solution when in contact with the L-PRF exudate.

Oral prebiotics and the influence of environmental conditions in vitro.

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.

In vitro Increased Respiratory Activity of Selected Oral Bacteria May Explain Competitive and Collaborative Interactions in the Oral Microbiome.

Understanding the driving forces behind the shifts in the ecological balance of the oral microbiota will become essential for the future management and treatment of periodontitis. As the use of competitive approaches for modulating bacterial outgrowth is unexplored in the oral ecosystem, our study aimed to investigate both the associations among groups of functional compounds and the impact of individual substrates on selected members of the oral microbiome. We employed the Phenotype Microarray high-throughput technology to analyse the microbial cellular phenotypes of 15 oral bacteria. Multivariate statistical analysis was used to detect respiratory activity triggers and to assess similar metabolic activities. Carbon and nitrogen were relevant for the respiration of health-associated bacteria, explaining competitive interactions when grown in biofilms. Carbon, nitrogen, and peptides tended to decrease the respiratory activity of all pathobionts, but not significantly. None of the evaluated compounds significantly increased activity of pathobionts at both 24 and 48 h. Additionally, metabolite requirements of pathobionts were dissimilar, suggesting that collective modulation of their respiratory activity may be challenging. Flow cytometry indicated that the metabolic activity detected in the Biolog plates may not be a direct result of the number of bacterial cells. In addition, damage to the cell membrane may not influence overall respiratory activity. Our methodology confirmed previously reported competitive and collaborative interactions among bacterial groups, which could be used either as marker of health status or as targets for modulation of the oral environment.

Biofilm-induced changes to the composite surface.

OBJECTIVES: Composites may undergo biodegradation in the oral cavity. The objective was to investigate the effect of single- and multi-species biofilms on the surface roughness and topography of two composites. METHODS: Disk-shaped specimens of a paste-like, Bis-GMA-free (Gradia Direct Anterior, GC), and a flowable, Bis-GMA-based composite (Tetric EvoFlow, Ivoclar-Vivadent) were prepared. After ethylene-oxide sterilization (38°C), specimens (n=3) were incubated with Streptococcus mutans or mixed bacterial culture (Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum). As negative controls, unexposed specimens and specimens exposed to sterile medium (BHI) were used. Specimens exposed to acidified BHI medium (pH=5) and enzymatic solution of cholesterol esterase served as positive control. Following 6-week incubation, the attached biofilms were collected for real-time PCR assessment, after which the surface roughness and topography of the specimens were analyzed with atomic force microscopy. Surface hydrophilicity/hydrophobicity was determined by contact angle measurements. Biofilm structure was analyzed with scanning electron microscopy. RESULTS: Even though multi-species biofilms were thicker, with more cells attached, they did not significantly affect the surface roughness of the composites. On the other hand, S. mutans alone significantly increased the roughness of Tetric by 40.3%, while its effect on Gradia was lower (12%). The total amount of attached bacteria, however, did not differ between the composites. CONCLUSIONS: S. mutans can increase the surface roughness of composites, depending on their composition. This ability of S. mutans is, however, mitigated in co-culture with other species. In particular, bacterial esterases seem to be responsible for the increased composite surface roughness upon biofilms exposure. CLINICAL SIGNIFICANCE: Cariogenic bacteria can degrade composites, thereby increasing the surface roughness. Increased roughness and subsequent improved bacterial accumulation may facilitate the development of secondary caries around composites, which is the most common reason for the restoration failure.

Necrotrophic growth of periodontopathogens is a novel virulence factor in oral 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.

Nutritional stimulation of commensal oral bacteria suppresses pathogens: the prebiotic concept.

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 by neutralizing commensal mediated inhibition of pathobionts.

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.

Antimicrobial effects of commensal oral species are regulated by environmental factors.

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 24h 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.

The effect of a streptococci containing probiotic in periodontal therapy: a randomized controlled trial.

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.

Effect of Bdellovibrio bacteriovorus HD100 on multispecies oral communities.

The predation of Bdellovibrio bacteriovorus on different periodontal pathogens has already been described. However, it is necessary to consider the polymicrobial nature of periodontal disease. The current study explores the predation of Bdellovibrio on oral pathogens organized in multispecies communities. The effect of the predator was evaluated on in vitro six species communities with microbial culturing. Additionally, the effect on ex vivo subgingival plaque and saliva samples from periodontitis patients was assessed. In the latter experiment results were examined with microbial culturing, quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). The latter technique was used to get an overview of the whole mixed microbial population. Results showed that even in more complex models, B. bacteriovorus was still able to predate on Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. However predation on Prevotella intermedia and Porphyromonas gingivalis could not be validated in multispecies models. The effect of Bdellovibrio was not restricted to the target bacteria. Changes in the overall ecology of the different models were evident. It could be concluded that the efficiency of predation decreased when complexity of the models increased. However, B. bacteriovorus was able to attack two important oral pathogens, F. nucleatum, and A. actinomycetemcomitans, even when present in ex vivo clinical samples. These effects still have to be validated in in vivo models to see the impact of Bdellovibrio on the whole bacterial ecology.

Probiotics reduce mutans streptococci counts in humans: a systematic review and meta-analysis.

OBJECTIVES: Systematically review the available literature regarding the caries-preventive effect of probiotics. DATA, SOURCES AND STUDY SELECTION: An electronic search was conducted in three databases (PubMed MEDLINE, ISI Web of Science and Cochrane Library) to identify all suitable studies. The outcomes had to be presented as the effect of probiotics on the incidence of caries or on the levels of mutans streptococci and/or Lactobacillus species. Human studies, written in English, with at least 15 participants, comparing a probiotic product with a placebo/no probiotic were included. Where possible, a meta-analysis was performed to obtain quantitative data. RESULTS: Since only two articles presented useful data on the caries incidence, we focused on the surrogate endpoints: mutans streptococci and/or Lactobacillus counts. The meta-analysis showed that when the probiotic and control group are compared after treatment, significantly more patients in the probiotic group had low mutans streptococci (<10(5) CFU/ml) counts and significantly less patients had high (>10(6) CFU/ml) counts. Regarding the Lactobacillus counts, comparing the probiotic and control group at the end of the probiotic use, no significant differences could be observed, neither in low (<10(4) CFU/ml) nor in high Lactobacillus (>10(6) CFU/ml) counts. CONCLUSIONS: Within the limitations of the available data, it may be concluded that probiotics decrease the mutans streptococci counts. This suggests that probiotics could have a positive effect in the prevention of caries. CLINICAL RELEVANCE: There is insufficient evidence that probiotics can prevent caries, but they can reduce the mutans streptococci counts.