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Concept: Oral microbiology

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BACKGROUND: The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans. RESULTS: Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r2 >= 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 mul sample in a final volume of 10 mul on the Light Cycler 480. CONCLUSIONS: In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment.

Concepts: DNA, Bacteria, Polymerase chain reaction, Microbiology, Pathogen, DNA replication, DNA polymerase, Oral microbiology

27

Purpose: To assess and compare the antimicrobial potential and determine the minimum inhibitory concentration (MIC) of Jasminum grandiflorum and Hibiscus rosa-sinensis extracts as potential anti-pathogenic agents in dental caries. Materials and Methods: Aqueous and ethanol (cold and hot) extracts prepared from leaves of Jasminum grandiflorum and Hibiscus rosa-sinensis were screened for in vitro antimicrobial activity against Streptococcus mutans and Lactobacillus acidophilus using the agar well diffusion method. The lowest concentration of every extract considered as the minimum inhibitory concentration (MIC) was determined for both test organisms. Statistical analysis was performed with one-way analysis of variance (ANOVA). Results: At lower concentrations, hot ethanol Jasminum grandiflorum (10 μg/ml) and Hibiscus rosa-sinensis (25 μg/ml) extracts were found to have statistically significant (P ≤ 0.05) antimicrobial activity against S. mutans and L. acidophilus with MIC values of 6.25 μg/ml and 25 μg/ml, respectively. A proportional increase in their antimicrobial activity (zone of inhibition) was observed. Conclusion: Both extracts were found to be antimicrobially active and contain compounds with therapeutic potential. Nevertheless, clinical trials on the effect of these plants are essential before advocating large-scale therapy.

Concepts: Statistics, Statistical significance, Dental caries, Analysis of variance, Lactobacillus, Lactobacillus acidophilus, Streptococcus mutans, Oral microbiology

27

The present clinical study aimed to investigate if there are differences in microbiological outcomes dependent on the subgingival biofilm collection method. Subgingival biofilm samples were collected from the four deepest pockets (>5 mm) of 17 patients with aggressive periodontitis (AgP) and 33 patients with chronic periodontitis (CP), first by paper point and thereafter by curette. Samples obtained with the same method were pooled together from each patient and forwarded for molecular microbiological analysis by a commercially available assay (IAI Pado Test 4.5) that estimates total bacterial load and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans. Data analysis included frequency of detection, quantification and correlation of detection levels between the two sampling methods. P. gingivalis, T. forsythia and T. denticola were detected in >90% of the samples, and their detection levels exhibited a strong correlation between sampling methods. The detection consistency of A. actinomycetemcomitans was 56% between the two sampling methods. A. actinomycetemcomitans was more readily detected by paper point compared with curette collection with a stronger correlation between the two methods in AgP. Subgingival biofilm sampling by curette or paper point does not yield differences in the detection of the three ‘red complex’ species. However, A. actinomycetemcomitans was more consistently detected by means of paper point collection, which can be crucial in the decision to administer antibiotics as an adjunctive periodontal treatment.

Concepts: Bacteria, Microbiology, Periodontology, Periodontitis, Dentistry, Porphyromonas gingivalis, Oral microbiology, Aggregatibacter actinomycetemcomitans

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The aim of the present study was to analyze the occurrence of Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia (T. forsythia), Treponema denticola (T. denticola), and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in patients with diabetes.

Concepts: Porphyromonas gingivalis, Oral microbiology, Aggregatibacter actinomycetemcomitans

27

Biomaterials made of biodegradable poly(α-hydroxyesters) like poly(lactide-co-glycolide) (PLGA) are known to decrease the pH in the vicinity of the implants. Bioactive glass (BG) is being investigated as a counteracting agent buffering the acidic degradation products. However, in dentistry the question arises whether an antibacterial effect is rather obtained from pure PLGA or from BG/PLGA composites, as BG has been proved antimicrobial. In the present study the antimicrobial properties of electrospun PLGA and BG45S5/PLGA fibres were investigated using human oral bacteria (specified with mass spectrometry) incubated for up to 24 h. BG45S5 nanoparticles were prepared by flame spray synthesis. The change in colony-forming units (CFU) of the bacteria was put into correlation with the pH of the medium during incubation. Morphology and structure of the scaffolds as well as the appearance of the bacteria were followed by microscopy. Additionally, we studied if the presence of BG45S5 had an influence on the degradation speed of the polymer. Finally, it turned out that the pH increase induced by BG45S5 presence in the scaffold did not last long enough to show a reduction in CFU. On the contrary, pure PLGA demonstrated antibacterial properties that should be taken into consideration when designing biomaterials for dental applications.

Concepts: Bacteria, Mass spectrometry, Microbiology, Dental implant, Antimicrobial, Oral microbiology, Scaffolding, The Medium

27

The prevalence of dental caries continues to increase and novel strategies to reverse this trend appear necessary. The probiotic Streptococcus salivarius strain M18 offers potential to confer oral health benefits since it produces a) bacteriocins targeting the important cariogenic species Streptococcus mutans and b) the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification respectively. In a randomised double-blind, placebo-controlled study of 100 dental caries- active children, treatment with strain M18 was administered for three months and the participants were assessed for changes to their plaque score, gingival and soft tissue health and to their salivary levels of S. salivarius, Streptococcus mutans, lactobacilli, beta haemolytic streptococci and Candida species. At treatment end, the plaque scores were significantly (P=0.05) lower for children in the M18-treated group especially in subjects having high initial plaque scores. The absence of any significant adverse events supported the safety of the probiotic treatment. Cell culture analyses of sequential saliva samples showed no differences between the probiotic and placebo groups in the counts of the specifically-enumerated oral microorganisms, with the exception of that subgroup of the M18-treated children who appeared to have colonised most effectively with M18. This subgroup exhibited reduced S. mutans counts, indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the efficiency of colonisation is increased. It is concluded that S. salivarius M18 can provide oral health benefits when taken regularly.

Concepts: Streptococcus, Dental caries, Lactobacillus, Dental plaque, Streptococcaceae, Streptococcus mutans, Oral microbiology, Streptococcus salivarius

21

Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

Concepts: Amino acid, Streptococcus, Dental caries, Dental plaque, Tooth enamel, Sucrose, Streptococcus mutans, Oral microbiology

21

Streptococcus mutans employs a key virulence factor, three glucosyltransferase (GtfBCD) enzymes to establish cariogenic biofilms. Therefore, the inhibition of GtfBCD would provide anti-virulence therapeutics. Here a small molecule library of 500,000 small molecule compounds was screened in silico against the available crystal structure of the GtfC catalytic domain. Based on the predicted binding affinities and drug-like properties, small molecules were selected and evaluated for their ability to reduce S. mutans biofilms, as well as inhibit the activity of Gtfs. The most potent inhibitor was further characterized for Gtf binding using OctetRed instrument, which yielded low micromolar KD against GtfB and nanomolar KD against GtfC, demonstrating selectivity towards GtfC. Additionally, the lead compound did not affect the overall growth of S. mutans and commensal oral bacteria, and selectively inhibit the biofilm formation by S. mutans, indicative of its selectivity and non-bactericidal nature. The lead compound also effectively reduced cariogenicity in vivo in a rat model of dental caries. An analog that docked poorly in the GtfC catalytic domain failed to inhibit the activity of Gtfs and S. mutans biofilms, signifying the specificity of the lead compound. This report illustrates the validity and potential of structure-based design of anti-S. mutans virulence inhibitors.

Concepts: Bacteria, Enzyme, Enzyme inhibitor, Inhibitor, Dental caries, Dental plaque, Streptococcus mutans, Oral microbiology

20

Understanding how the body’s natural defenses function to protect the oral cavity from the myriad of bacteria that colonize its surfaces is an ongoing topic of research that can lead to breakthroughs in treatment and prevention. One key defense mechanism on all moist epithelial linings, such as the mouth, gastrointestinal tract and lungs, is a layer of thick, well-hydrated mucus. The main gel-forming component of mucus are mucins, large glycoproteins that play a key role in host defense. This study focuses on elucidating the connection between MUC5B salivary mucins and dental caries, one of the most common oral diseases. Dental caries are predominantly caused by Streptococcus mutans adherence and biofilm formation on the tooth surface. Once S. mutans adheres to the tooth, it produces organic acids as metabolic byproducts that dissolve tooth enamel, leading to cavity formation. We utilize colony forming units and fluorescent microscopy to quantitatively show that S. mutans attachment and biofilm formation is most robust in the presence of sucrose and that aqueous solutions of purified human MUC5B protect surfaces by acting as an anti-biofouling agent in the presence of sucrose. In addition, we find that MUC5B does not alter S. mutans growth and decreases surface attachment and biofilm formation by maintaining S. mutans in the planktonic form. These insights point to the importance of salivary mucins in oral health and lead to a better understanding of how MUC5B could play a role in cavity prevention or diagnosis.

Concepts: Streptococcus, Lactic acid, Dental caries, Dental plaque, Tooth enamel, Sucrose, Streptococcus mutans, Oral microbiology

2

Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

Concepts: Bacteria, Biofilm, Dental plaque, Tooth enamel, Streptococcus mutans, Oral microbiology, Glass ionomer cement, Dental materials