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 multispecies 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

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

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

Nutrient mediated modulation of the oral microbiota

Periodontal diseases are a significant public health burden, of which severe periodontitis is the 6th-most prevalent disease, affecting 11% of the global population. Periodontitis is defined as infections of the tooth supporting tissue, leading to destruction of the connective tissue and alveolar bone, eventually resulting in tooth loss. While dental biofilm are known to play a major role in the development of periodontal diseases, the exact microbial etiology is still unknown. The ecological plaque hypothesis states that the host, the environment, the presence of periodontal pathogens and the absence of beneficial bacteria are the most important factors in the development of periodontal diseases. The shift from a homeostatic microbial composition towards a dysbiotic microbiota in a susceptible host will lead to the development of periodontitis. To date, standard periodontal therapy focuses mainly on the reduction and elimination of the bacterial load with subsequent improvement in clinical parameters. However, fast bacterial translocation and recolonization increases the risk of recurrence of disease, making maintenance therapy a lifelong requirement for the patients. One new treatment strategy focuses on controlling recolonization by means of probiotic bacteria. Although the use of certain probiotic bacteria resulted in an improvement in clinical parameters as well as in a delayed recolonization by pathogenic bacteria, the colonization of probiotic bacteria in the oral cavity is only temporary. Therefore, the stimulation of the protective and beneficial properties of the natural resident microbiota might reveal a new treatment approach. The prebiotic concept has already been introduced in gastrointestinal health. By means of specific food ingredients, selective changes in the composition or activity of the indigenous gastrointestinal are induced, thus conferring health benefits to the host. The main aim of this thesis was to investigate if the prebiotic concept can be transferred to oral health to reveal an alternative treatment approach for periodontitis. To identify potential prebiotic compounds for oral health, Phenotype MicroArrays were used as a high-throughput method to monitor the change in respiratory activity of 16 oral bacteria in response to 759 nutritional compounds. Multivariate statistical analysis was employed to investigate metabolic associations within and among bacterial groups, analyzing collaborative or competitive interactions of bacteria. Further, compounds were selected based on their selective stimulation of individual bacteria in metabolic activity, growth and biofilm formation. Seven compounds were subsequently used in dual species competition assays of beneficial and pathogenic bacteria. N-acetyl-D-mannosamine, L-arginine, and beta-methyl-D-galactoside selectively stimulated beneficial oral bacteria, leading to a suppression of pathogenic species. Hence, mixed species biofilm communities were shifted towards a composition dominated by beneficial oral bacteria at in vitro level. To validate the selective stimulatory effect of beneficial bacteria, a chemostat culture containing 14 model bacteria was developed. Nine potential prebiotic substrates were tested on these multispecies biofilms, and biofilm composition was analyzed by vitality qPCR. N-acetyl-D-mannosamine, succinic acid, and the di-peptide Met-Pro were able to stimulate the beneficial proportion of the biofilm to above 95%. Additionally, the effect of environmental factors, such as pH, nutrient availability, oxygen concentration and prebiotic dose, on the efficacy of the prebiotic substances was evaluated. Throughout the experiments, N-acetyl-D-mannosamine could be identified as the most promising prebiotic compound, shifting the biofilm composition despite changing conditions towards a beneficial dominated community of 97%. In addition, the potential of N-acetyl-D-mannosamine to increase the resistance of established beneficial biofilms to invading pathogens was analyzed. Incorporation of pathogenic species was significantly decreased when compared to the control treatment, thus, possibly enabling the use of prebiotics to control or prevent the translocation and recolonization of pathogenic bacteria in the oral cavity. Although the exact mechanism of the prebiotic treatment needs to be investigated, data indicated that N-acetyl-D-mannosamine also interferes with the co-aggregation of beneficial and pathogenic bacteria. The prebiotic concept shows promising potential as a new treatment approach for periodontal diseases by shifting the bacterial composition of mixed biofilm communities towards a health associated microbiota and preventing the incorporation of pathogenic bacteria into established biofilms.status: publishe

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(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(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.status: publishe

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.status: publishe

Lack of Buffering by Composites Promotes Shift to More Cariogenic Bacteria

Secondary caries (SC) remains a very important problem with composite restorations. The objectives of this study were to test the acid-buffering ability of several restorative materials and to evaluate whether buffering of the restorative material has an impact on the microbial composition of the biofilm. Disk-shaped specimens of conventional composite, composite with surface prereacted glass-ionomer filler particles (so-called giomer), glass-ionomer cement (GIC), amalgam, and hydroxyapatite (HAp) (control) were exposed to aqueous solutions with pH 4, 5, 6, and 7 and to the medium containing bacteria-produced acids, and pH changes were recorded over several days. Next, material specimens were immersed in bacterial growth medium with pH adjusted to 5. After a 24-h incubation, the extracts were collected and inoculated with a cariogenic (Streptococcus mutans) and a noncariogenic (Streptococcus sanguinis) species. The bacterial growth was monitored both in a single-species model by spectrophotometry and in a dual-species model by viability quantitative polymerase chain reaction. Amalgam and HAp showed the strongest acid-buffering ability, followed by the GIC and the giomer, while the conventional composite did not exhibit any buffering capacity. Furthermore, due to the lack of acid-buffering abilities, composite was not able to increase the pH of the medium (pH 5), which, in the absence of antibacterial properties, allowed the growth of S. mutans, while the growth of S. sanguinis, a less aciduric species, was completely inhibited. A similar effect was observed when bacteria were cultured together: there was a higher percentage of S. mutans and lower percentage of S. sanguinis with the conventional composite than with other materials and HAp. In conclusion, conventional composites lack the ability to increase the local pH, which leads to the outgrowth of more acidogenic/aciduric bacteria and higher cariogenicity of the biofilm. Together with lack of antibacterial properties, lack of buffering may account for the higher susceptibility of composites to SC.status: publishe

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 F. nucleatum and A. actinomycetemcomitans. However predation on P. intermedia and P. 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.publisher: Elsevier articletitle: Effect of Bdellovibrio bacteriovorus HD100 on multispecies oral communities journaltitle: Anaerobe articlelink: http://dx.doi.org/10.1016/j.anaerobe.2014.09.011 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

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.status: publishe