Martine Pauwels

Initial Subgingival Colonization of ‘Pristine’ Pockets

The treatment of periodontitis/peri-implantitis involves the reduction/eradication of periopathogens. After therapy, beneficial and pathogenic species recolonize the subgingival area. The dynamics of recolonization and especially the role of the supragingival environment in this process are still not well-understood. This prospective, split-mouth study followed the early colonization of ‘pristine’ pockets created during implant surgery (16 partially edentulous patients), to record the time needed before a complex subgingival flora could be established with the supragingival area as the single source. Four subgingival plaque samples were taken from shallow and medium pockets around implants (test), and neighboring teeth (undisturbed microbiota as reference) 1, 2, and 4 wks after abutment connection. Checkerboard DNA-DNA hybridization and culture data revealed a complex microbiota (including several pathogenic species) in the pristine pockets within a wk, with a minimal increase in counts up to 4 wks. Analysis of these data demonstrated that, even with the supragingival environment as the single source for colonizing bacteria, a complex subgingival microbiota can develop within 1 wk.

Live/dead real-time polymerase chain reaction to assess new therapies against dental plaque-related pathologies

DNA-based methodology for the identification and detection of specific bacteria in dental plaque offers advantages over culturing techniques. One drawback of current molecular techniques like real-time quantitative polymerase chain reaction (RT-QPCR) is that they are not able to distinguish between live or dead bacteria. To overcome this problem an assay was assessed to discriminate between viable or dead bacteria using DNA intercalating substances, propidium monoazide (PMA) and ethidium monoazide (EMA) in combination with RT-QPCR. The assay was tested on oral pathogens: Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. To determine the effectiveness of EMA and PMA, different concentrations (from 5 to 100 μg ml(-1)) of the substances were added to viable or heat-killed suspensions of both organisms (ranging from 10(8) to 10(4) colony-forming units ml(-1)). Afterwards, PMA was tested on mixtures of varying ratios of viable and dead cells. After DNA extraction, RT-QPCR was performed using species-specific primers. Both compounds inhibited PCR amplification from dead cells. The EMA treatment resulted in the largest signal decrease but EMA also inhibited DNA amplification from viable cells. For this reason, PMA was selected for use in further experiments. It was shown to be efficient in allowing selective PCR detection of only viable cells in mixtures containing both viable and dead cells. The amount of amplified DNA corresponded to the percentage of viable cells in the sample. The developed assay will potentially be useful for assessing bacterial loads remaining after disinfection protocols without interference by non-viable bacteria.

Microbial adhesion on different bracket types in vitro.

OBJECTIVE To test the hypothesis that there are differences in total bacterial counts and capacity for biofilm formation between seven different bracket types. MATERIAL AND METHODS By means of an in vitro experiment, seven commercially available bracket systems (Damon [A], Clarity [B], Mystique [C], Speed [D], Victory MBT [E], Micro-loc [F], and Generus [G]) were compared. A total of 25 premolar brackets of each bracket system were incubated in brain heart infusion medium containing the saliva and bacteria of two orthodontic patients. After 72 hours, the amounts of aerobe and anaerobe bacteria were determined by counting the colony-forming units (CFU). The CFU ratio (aerobe/anaerobe) also was calculated, and the black pigmented bacteria were analyzed. RESULTS Significant differences between the different bracket types in terms of biofilm formation were found. Bracket types can be arbitrarily divided into low, intermediate, and high plaque-retaining brackets. The group with low adhesion consists of bracket types E, F, and G; the group with high adhesion of bracket types A, B, and C; and type D exhibits intermediate adhesion. The group with high microbial adhesion (A, B, and C) did present significantly lower CFU ratios (aerobe/anaerobe) than were exhibited by the other bracket systems (P < .05). CONCLUSION The hypothesis is accepted. Orthodontic brackets serve as different loci for biofilm formation; in this in vitro study, significant differences were noted between the different types of brackets.

Increase in Cariogenic Bacteria after Initial Periodontal Therapy

This study examined the hypothesis of an intra-oral shift, during initial periodontal therapy, from a periopathogenic to a cariogenic flora. Seventy-one patients with periodontitis were randomly allocated to one of the following treatment strategies: (1) scaling and root planing, quadrant by quadrant, at two-week intervals (NC); (2) full-mouth scaling and root planing within 24 hrs (FRP); or (3) full-mouth disinfection within 24 hrs, including antiseptics [chlorhexidine (CHX) or amine fluoride/stannous fluoride (F) for 2 mos, or CHX for 2 mos followed by F for 6 mos (CHX+F)]. At baseline and after 2, 4, and 8 mos, bacterial samples were taken from supra- and subgingival plaque, saliva, and tongue. The detection frequencies and relative proportions of Streptococcus mutans increased in the NC and FRP groups, but decreased in the F group. In the CHX group, these species disappeared temporarily, but they disappeared for the entire 8 mos in the CHX+F group. These observations were similar for all sample locations. The periopathogens decreased in all groups. This finding confirms the abovementioned hypothesis and indicates a need for caries prophylactic regimens.

Bacterial Antagonism Against Periodontopathogens

BACKGROUND The aim of the current study is to compare the prevalence of commensal bacteria, with beneficial properties, for healthy and diseased individuals and additionally to examine the inhibitory effect of some commercial dietary probiotics on periodontopathogens, comparing this inhibitory effect to that of orally derived beneficial bacteria. METHODS Subgingival plaque samples from 35 patients (healthy and periodontitis patients) were analyzed. Growth inhibition of the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans was examined using the agar overlay technique and agar well diffusion method. The quantification of the inhibitory effect was checked with the agar well diffusion method. RESULTS Using the agar overlay technique, the prevalence of strains antagonistic toward P. gingivalis, A. actinomycetemcomitans, and F. nucleatum was found to be higher in healthy individuals than in individuals with periodontitis, but this could not be validated by the agar well diffusion assay. Compared with the antagonistic activity of the isolated strains, the probiotic strains overall showed a stronger inhibition of the periodontal pathogens. CONCLUSION It was shown that some oral bacteria can cause antagonism toward periodontopathogens, and these observations underline the therapeutic potential of applications that stimulate oral health by the application of beneficial effector strains.

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.

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.