Vera Slomka

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.

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.

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.

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.

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.

Clinical concentrations of peroxidases cause dysbiosis in in vitro oral biofilms

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 (H2 O2 ) 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 H2 O2 . Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to 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.