Subgingival Biofilm Formation Research Articles

The Impact of Candida albicans in the Development, Kinetics, Structure, and Cell Viability of Biofilms on Implant Surfaces-An In Vitro Study with a Validated Multispecies Biofilm Model.

This study aimed to evaluate the impact of Candida albicans on subgingival biofilm formation on dental implant surfaces. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to compare biofilm structure and microbial biomass in the presence and absence of the fungus after periods of 24, 48, and 72 h. Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total micro-organisms for each of the biofilm-forming strains. A general linear model was applied to compare CLSM and qPCR results between the control and test conditions. The biofilm developed with C. albicans at 72 h had a higher bacterial biomass and a significantly higher cell viability (p < 0.05). After both 48 and 72 h of incubation, in the presence of C. albicans, there was a significant increase in counts of Fusobacterium nucleatum and Porphyromonas gingivalis and in the cell viability of Streptococcus oralis, Aggregatibacter actinomycetemcomitans, F. nucleatum, and P. gingivalis. Using a dynamic in vitro multispecies biofilm model, C. albicans exacerbated the development of the biofilm grown on dental implant surfaces, significantly increasing the number and cell viability of periodontal bacteria.

Metabolic activity of hydro-carbon-oxo-borate on a multispecies subgingival periodontal biofilm: a short communication

ObjectiveThis study evaluated the metabolic activity of hydro-carbon-oxo-borate complex (HCOBc) on a multispecies subgingival biofilm as well as its effects on cytotoxicity.Materials and methodsThe subgingival biofilm with 32 species related to periodontitis was formed in the Calgary Biofilm Device (CBD) for 7 days. Two different therapeutic schemes were adopted: (1) treatment with HCOBc, 0.12% chlorhexidine (CHX), and negative control group (without treatment) from day 3 until day 6, two times a day for 1 min each time, totaling 8 treatments and (2) a 24-h treatment on a biofilm grown for 6 days. After 7 days of formation, biofilm metabolic activity was determined by colorimetry assay, and bacterial counts and proportions of complexes were determined by DNA-DNA hybridization. Both substances’ cytotoxicity was evaluated by cell viability (XTT assay) and clonogenic survival assay on ovary epithelial CHO-K1 cells and an osteoblast precursor from calvaria MC3T3-E1 cells.ResultsThe first treatment scheme resulted in a significant reduction in biofilm’s metabolic activity by means of 77% by HCOBc and CHX treatments versus negative control. The total count of 11 and 25 species were decreased by treatment with hydro-carbon-oxo-borate complex and CHX, respectively, compared with the group without treatment (p < 0.05), highlighting a reduction in the levels of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Fusobacterium periodontium. CHX significantly reduced the count of 10 microorganisms compared to the group treated with HCOBc (p < 0.05). HCOBc and CHX significantly decreased the pathogenic red-complex proportion compared with control-treated biofilm, and HCOBc had even a more significant effect on the red complex than CHX had (p ≤ 0.05). For the second treatment scheme, HCOBc complex and CHX significantly decreased 61 and 72% of control biofilms’ metabolic activity and the counts of 27 and 26 species, respectively. HCOBc complex did not significantly affect the proportions of formed biofilms, while CHX significantly reduced red, orange, and yellow complexes. Both substances exhibited similar cytotoxicity results.ConclusionsThis short communication suggested that the HCOBc complex reduced a smaller number of bacterial species when compared to chlorhexidine during subgingival biofilm formation, but it was better than chlorhexidine in reducing red-complex bacterial proportions. Although HCOBc reduced the mature 6-day-old subgingival multispecies biofilms, it did not modify bacterial complexes’ ratios as chlorhexidine did on the biofilms mentioned above. Future in vivo studies are needed to validate these results.Clinical relevanceHCOBc complex could be used to reduce red-complex periodontal bacterial proportions.

Efficacy of ozonated water mouthwash on early plaque formation and gingival inflammation: a randomized controlled crossover clinical trial.

To evaluate the effect of ozonated water in early plaque formation and gingival inflammation. This was a randomized, controlled, double-blind, crossover clinical trial with two experimental periods of 96h each, with 10 washout days between them. The sample consisted of 42 dental students divided into Test Group, mouthwash of ozonated water, and Control Group, bidistilled water mouthwash. The participants were instructed not to perform oral hygiene and used the assigned mouthwash under supervision once a day. For the investigation of the initial subgingival biofilm formation, the Plaque Free Zone Index was used at 24, 48, 72, and 96h. The volume of gingival crevicular fluid, a questionnaire for taste perception assessment, and analysis of the adverse effects were also carried out. The percentage of conversion scores 0 and 1 to 2 of PFZ Index, the main outcome, for all dental surfaces showed no statistical difference between Test and Control groups, with 19.07 and 19.79, respectively. Also, there was not a significant difference in the score frequencies at each time point. Evaluation of GCF demonstrated that both groups had an increase in volume during experimental periods and that there was no statistically significant difference among groups. Test group had worse evaluation of taste perception and more adverse effects. Ozonated water seems not to affect the formation of supra and subgingival biofilms, as well as gingival inflammation. Mouthwash with ozonated water once a day do not affect supra and subgingival biofilm formation.

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro.

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P < 0.05) in biofilm formation occurred after 15min exposure to 20µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15min exposure to 20µM of LFchimera compared to control and 20-50µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.

Chlorhexidine with or without alcohol against biofilm formation: efficacy, adverse events and taste preference.

In recent years, different chlorhexidine formulations have been tested, including an alcohol-free alternative, but the effect of this solution on early biofilm formation is not clear. A crossover, randomized, double-blind clinical trial was conducted to evaluate the effect of two chlorhexidine solutions against supra- and subgingival biofilm formation (NCT#02656251). Thirty-five participants were randomized and asked to rinse twice daily with 15 ml of an alcohol-containing 0.12% chlorhexidine solution, an alcohol-free 0.12% chlorhexidine solution, or placebo. The study was conducted in three experimental periods of 4 days each, with a 10-day washout between the periods. All the experimental periods followed the same protocol, except that the solutions were switched. Biofilm distribution was evaluated every 24 hours by the Plaque-Free Zone Index, during 96 hours. Adverse events were self-reported and sensory evaluation was performed using a hedonic scale. Compared to the placebo, the chlorhexidine solutions resulted in a significantly higher number of surfaces free of plaque over 96 hours (p < 0.01), and were able to prevent subgingival biofilm formation (p < 0.01). The alcohol-free chlorhexidine solution was associated with a lower incidence of adverse events, compared with alcohol-containing chlorhexidine (p < 0.05); it also received better sensory evaluation and acceptance by trial participants, compared with the alcohol-containing chlorhexidine (p = 0.007), and had a similar inhibitory effect on the formation of supra- and subgingival biofilms.

Efficacy of a triclosan formula in controlling early subgingival biofilm formation: a randomized trial.

The aim of this study was to determine the efficacy of rinses with slurries of a dentifrice containing triclosan (TCS), as compared with rinses with slurries from a control dentifrice, in controlling early subgingival biofilm formation. A double-blind, randomized and cross-over clinical trial was designed, and 26 dental students were included. In the first period, participants were randomized to rinse with a TCS slurry or a control slurry, in a 12 h interval, and to refrain from mechanical cleaning. A Plaque Free Zone Index was assessed at 24 h, 48 h, 72 h and 96 h. After a washout period of 10 days, the second experimental period was conducted, following the same protocol as the first period, except that the slurry groups were switched. Use of the TCS slurry resulted in a significantly higher percentage of plaque-free surfaces, both at 24 h and at 72 h (p < 0.01). In the of 48-72 h interval, the triclosan slurry showed a lower percentage of sites converted to a score of 2 (38.1% for the test versus 40% for the control product, p = 0.015). In conclusion, rinsing with slurries of dentifrice containing TCS retards the down growth of bacterial biofilms from the supra- to the subgingival environment.

Effects of Streblus asper leaf extract on the biofilm formation of subgingival pathogens

Streblus asper leaf extract (SAE) possesses antibacterial activity towards Streptococcus mutans and periodontal pathogens, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. However, there is no report on inhibitory effects of SAE against subgingival biofilm formation. The purpose of this study was to investigate the inhibitory effects of SAE on subgingival biofilm formation using an in vitro model. Subgingival plaque samples from 5 periodontitis patients were cultivated in saliva-coated 96-well microtiter plates in the presence of SAE at concentrations 0.5–90mg/ml under anaerobic atmosphere at 37°C for 2, 4 and 8days by replacing every 2days with fresh medium alone or SAE-containing medium. Biofilm formation was determined quantitatively by crystal violet staining. TaqMan-based real-time polymerase chain reaction was used to quantify the numbers of P. gingivalis, A. actinomycetemcomitans and total bacteria in the grown subgingival biofilm. The results showed that SAE exhibited more than 70% anti-biofilm activity at 90mg/ml. The reduction in cell numbers of P. gingivalis, A. actinomycetemcomitans and total bacteria in subgingival biofilm of test groups were observed compared to untreated control. These results revealed that SAE is able to inhibit in vitro subgingival biofilm formation and reduce the numbers of P. gingivalis, A. actinomycetemcomitans and total bacteria.

Strain-specific colonization patterns and serum modulation of multi-species oral biofilm development

Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.

RCT comparing minimally with moderately rough implants. Part 2: microbial observations

Most current implants have a moderately rough surface (compared with older minimally rough "turned" implants) to facilitate osseointegration. This randomized controlled trial (RCT), with split-mouth design, examined whether this increased surface roughness influenced the initial subgingival plaque formation. Ten fully edentulous and eight partially edentulous patients, all with a history of severe periodontitis, received 4-6 implants (mandible or maxilla). Per jaw, both minimally (turned) and moderately rough (TiUnite) implants (MKIII; Nobel Biocare) were alternated. Also, the healing and final abutments had similar surface characteristics. Subgingival biofilm formation was followed up for 1 year, and samples were analyzed by culture technique, qPCR and checkerboard Over the entire period, no statistically significant differences could be detected in subgingival microbiota between the minimally and moderately rough surfaces. In partially edentulous patients, the biofilm matured to a higher concentration of pathogens when compared with fully edentulous patients. The subgingival implant composition and concentration in partially edentulous patients were comparable to the subgingival microbiota along teeth. The roughness of the more modern implants did not influence the biofilm formation during the first year of implant loading.

Microbial diversity of supra- and subgingival biofilms on freshly colonized titanium implant abutments in the human mouth

Supra- and subgingival biofilm formation is considered to be mainly responsible for early implant failure caused by inflammations of periimplant tissues. Nevertheless, little is known about the complex microbial diversity and interindividual similarities around dental implants. An atraumatic assessment was made of the diversity of microbial communities around titanium implants by single strand conformation polymorphism (SSCP) analysis of the 16S rRNA gene amplicons as well as subsequent sequence analysis. Samples of adherent supra- and subgingival periimplant biofilms were collected from ten patients. Additionally, samples of sulcusfluid were taken at titanium implant abutments and remaining teeth. The bacteria in the samples were characterized by SSCP and sequence analysis. A high diversity of bacteria varying between patients and within one patient at different locations was found. Bacteria characteristic for sulcusfluid and supra- and subgingival biofilm communities were identified. Sulcusfluid of the abutments showed higher abundance of Streptococcus species than from residual teeth. Prevotella and Rothia species frequently reported from the oral cavity were not detected at the abutments suggesting a role as late colonizers. Different niches in the human mouth are characterized by specific groups of bacteria. Implant abutments are a very valuable approach to study dental biofilm development in vivo.

Subgingival biofilm formation

The human body contains numerous distinctive ecosystems that provide a unique environment for colonizing microorganisms. The periodontal pocket is one such microniche. This environment is partially sheltered from the physical shear forces in the oral cavity and contains the hard, nonshedding surfaces of the tooth root along with the shedding surfaces of gingival mucosa. The junctional epithelium, which is attached to the tooth root, is poorly differentiated, lacks keratinization and has relatively wide intercellular spaces. Consequently, junctional epithelium is permeable and allows the migration of polymorphonuclear leukocytes into the periodontal pocket. Furthermore, the tissues in the periodontal pocket are bathed in gingival crevicular fluid, a serum exudate with antioxidant properties. The initial bacterial colonizers attach to the available surfaces, as discussed elsewhere in this volume of Periodontology 2000. Later colonizers attach to the antecedent organisms and assemble into polymicrobial communities. The biofilms on the hard surfaces develop into spatially organized structures that can extend several hundred micrometers from the surface. By contrast, the epithelial surfaces, which are continually being sloughed and replenished, tend to be colonized with monolayers of microorganisms. However, several of the more pathogenic species of bacteria are able to invade the gingival cells and tissues where they can remain viable and thus constitute a nidus of infection. Interspecies adherence interactions help to shape the temporal and spatial development of the complex bacterial consortia in the gingival crevice. Bacteria within these communities encounter high cell densities and, in consequence, community living involves adaptation to higher (and unevenly distributed) levels of metabolic by-products, secondary metabolities and other secreted molecules, and to the sporadic availability of nutrients and oxygen. Bacterial inhabitants of biofilms are known to both collaborate (e.g. through nutritional cross-feeding) and compete (e.g. through production of bacteriocins) as they strive to optimize their adaptation to these environmental constraints. Bacteria can also communicate with one another through a variety of sensing and response systems based on either cell-to-cell contact or detection of soluble mediators. The signaling molecules are processed through transcriptional and post-transcriptional networks and they allow bacterial inhabitants of biofilms to coordinate activities at a group or community level. An understanding of the mechanisms of subgingival biofilm formation and development needs, therefore, to accommodate the multiple interspecies interactions that occur in polymicrobial communities.