Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Insights into preventive measures for dental erosion

Dental erosion is defined as the loss of tooth substance by acid exposure not involving bacteria. The etiology of erosion is related to different behavioral, biological and chemical factors. Based on an overview of the current literature, this paper presents a summary of the preventive strategies relevant for patients suffering from dental erosion. Behavioral factors, such as special drinking habits, unhealthy lifestyle factors or occupational acid exposure, might modify the extent of dental erosion. Thus, preventive strategies have to include measures to reduce the frequency and duration of acid exposure as well as adequate oral hygiene measures, as it is known that eroded surfaces are more susceptible to abrasion. Biological factors, such as saliva or acquired pellicle, act protectively against erosive demineralization. Therefore, the production of saliva should be enhanced, especially in patients with hyposalivation or xerostomia. With regard to chemical factors, the modification of acidic solutions with ions, especially calcium, was shown to reduce the demineralization, but the efficacy depends on the other chemical factors, such as the type of acid. To enhance the remineralization of eroded surfaces and to prevent further progression of dental wear, high-concentrated fluoride applications are recommended. Currently, little information is available about the efficacy of other preventive strategies, such as calcium and laser application, as well as the use of matrix metalloproteinase inhibitors. Further studies considering these factors are required. In conclusion, preventive strategies for patients suffering from erosion are mainly obtained from in vitro and in situ studies and include dietary counseling, stimulation of salivary flow, optimization of fluoride regimens, modification of erosive beverages and adequate oral hygiene measures.

Antimicrobial Activity of Diterpenes from Viguiera arenaria against Endodontic Bacteria

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14), 15-dien-19-oic acid, its sodium salt and ent-8(14), 15-pimaradien-3 beta-ol were the most active, displaying MIC values ranging from 1 to 10 mu g mL(-1). The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

In Vitro Adhesion of Streptococcus sanguinis to Dentine Root Surface After Treatment with Er:Yag Laser, Ultrasonic System, or Manual Curette

Objective: The purpose of this in vitro study was to evaluate the dentine root surface roughness and the adherence of Streptococcus sanguinis (ATCC 10556) after treatment with an ultrasonic system, Er:YAG laser, or manual curette. Background Data: Bacterial adhesion and formation of dental biofilm after scaling and root planing may be a challenge to the long-term stability of periodontal therapy. Materials and Methods: Forty flattened bovine roots were randomly assigned to one of the following groups: ultrasonic system (n = 10); Er:YAG laser (n = 10); manual curette (n = 10); or control untreated roots (n = 10). The mean surface roughness (Ra, mu m) of the specimens before and after exposure to each treatment was determined using a surface profilometer. In addition, S. sanguinis was grown on the treated and untreated specimens and the amounts of retained bacteria on the surfaces were measured by culture method. Results: All treatments increased the Ra; however, the roughest surface was produced by the curettes. In addition, the specimens treated with curettes showed the highest S. sanguinis adhesion. There was a significant positive correlation between roughness values and bacterial cells counts. Conclusion: S. sanguinis adhesion was the highest on the curette-treated dentine root surfaces, which also presented the greatest surface roughness.

Antimicrobial Activity of Terpenoids from Copaifera langsdorffii Desf. Against Cariogenic Bacteria

In the present work, the anticariogenic activities of nine labdane type-diterpenes and four sesquiterpenes were investigated. Among these metabolites, (-)-copalic acid (CA) was the most active compound displaying MIC values very promising (ranging from 2.0 to 6.0 mu g/mL) against the main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. Time kill assays performed with CA against the primary causative agent (S. mutans) revealed that, in the first 12 h, this compound only inhibits the growth of the inoculum (bacteriostatic effect). However, its bactericidal effect is clearly noted thereafter (between 12 and 24 h). Also, CA did not show a synergistic effect when combined with the anticariogenic gold standard (chlorhexidine, CHD) in the checkerboard assays against S. mutans. In conclusion, the results points out CA as an important metabolite in the search for new effective anticariogenic agents. Copyright (C) 2010 John Wiley & Sons, Ltd.

Bacterial Adhesion on Smooth and Rough Titanium Surfaces After Treatment With Different Instruments

Background: Newly formed biofilm after implant debridement may challenge the long-term stability of peri-implant therapy. This in vitro study aimed to assess the roughness and adherence of Streptococcus sanguinis after treatment of smooth and rough titanium surfaces with an erbium-doped: yttrium, aluminum, and garnet (Er:YAG) laser, metal and plastic curets, and an air-powder abrasive system. Methods: Forty titanium disks with smooth-machined surfaces and 40 with sand-blasted and acid-etched surfaces were divided into the following treatment groups: Er:YAG laser; plastic curet; metal curet, and air-powder abrasive system. The surface roughness (roughness average [Raj) before and after treatments was determined using a profilometer. S. sanguinis (American Type Culture Collection 10556) was grown on treated and untreated specimens, and the amounts of retained bacteria on the surfaces were measured by the culture method. Rough and smooth surfaces with and without a suspension of S. sanguinis were also analyzed using scanning electron microscopy (SEM). Results: For smooth surfaces, the roughest surfaces were produced by metal curets (repeated - measures analysis of variance [ANOVA] and Tukey test; P<0.05). The rough-surface profile was not altered by any of the treatments (repeated-measures ANOVA; P>0.05). Rough surfaces treated with metal curets and air-powder abrasion showed the lowest level of bacteria] adhesion (two-way ANOVA and Tukey test; P<0.05). SEM analysis revealed distinct surface profiles produced by all devices. Conclusions: Metal curets are not recommended for smooth titanium surface debridement due to severe texture alteration. Rough surfaces treated with a metal curet and the air-powder abrasive system were less susceptible to bacterial adhesion, probably due to texture modification and the presence of abrasive deposits. J Periodontol 2009;80: 1824-1832.

Detection of Herpesviruses and Periodontal Pathogens in Subgingival Plaque of Patients With Chronic Periodontitis, Generalized Aggressive Periodontitis, or Gingivitis

Background: Herpesviruses may be related to the etiology of aggressive periodontitis (AgP) and chronic periodontitis (CP) by triggering periodontal destruction or by increasing the risk for bacterial infection. This case-control study evaluated the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus type 1 (EBV-1), human cytomegalovirus (HCMV), Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia (previously T. forsythensis) in patients with generalized AgP (AgP group), CP (CP group), or gingivitis (G group) and in healthy individuals (C group). Methods: Subgingival plaque samples were collected with paper points from 30 patients in each group. The nested polymerase chain reaction (PCR) method was used to detect HSV-1, EBV-1, and HCMV. Bacteria were identified by 16S rRNA-based PCR. Results: HSV-1, HCMV, and EBV-1 were detected in 86.7%, 46.7%, and 33.3% of the AgP group, respectively; in 40.0%, 50.0%, and 46.7% of the CP group, respectively; in 53.3%, 40.0%, and 20.0% of the G group, respectively; and in 20.0%, 56.7%, and 0.0% of the C group, respectively. A. actinomycetemcomitans was detected significantly more often in the AgP group compared to the other groups (P<0.005). P. gingivalis and T. forsythia were identified more frequently in AgP and CP groups, and AgP, CP, and G groups had higher frequencies of P. intermedia compared to the C group. Conclusion: In Brazilian patients, HSV-1 and EBV-1, rather than HCMV, were more frequently associated with CP and AgP. J Periodontol 2008;79:2313-2321.

Clinical, Histological, and Microbiological Findings in Peri-Implant Disease: A Pilot Study

Objectives: This study is intended to verify the correlation among clinical indices of the peri-implant soft tissues, the histological condition and the presence of 3 pathogens commonly associated with peri-implant diseases (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia). Materials: Four clinical indices, Gingival Index (GI), Sulcus Bleeding Index, GI modified by Mombelli, and Plaque Index modified by Mombelli (mPI) were evaluated around I dental implant of each subject (n = 10). Subgingival plaque was collected for bacterial analysis (polymerase chain reaction) and a biopsy of peri-implant soft tissues for histological analysis was harvested. The clinical indices and detected pathogens correlated with a developed histological index (HI). Results: There was no statistically significant relationship between the clinical indices (GI, Sulcus Bleeding Index, and GI modified by Mombelli) and the HI, except for the mPI on the central area of lingual aspects (r = 0.85, P = 0.0029). There was a tendency for a positive correlation between the mPI on the central area of buccal aspects and the HI (r = 0.63, P = 0.0544). The counting of lymphocytes and plasmocytes correlated positively with 111, thus suggesting the index reliability. The prevalence of A. actinomycetemcomitans, P. gingivalis, and T. forsythia did not present a significant relationship with the HI. Conclusion: Despite the small number of samples and the poor statistical significance, the mPI seems to be useful for evaluation of inflammatory severity on soft tissue around dental implants as demonstrated by its relationship with the HI. Further studies are necessary to elucidate this subject. (Implant Dent 2009;18:334-344)

In vivo Acid Etching Effect on Bacteria within Caries-Affected Dentin

Acid etching procedures may disrupt residual bacteria and contribute to the success of incomplete caries removal followed by adhesive restoration. This study evaluated the in vivo effect of acid etching on cariogenic bacterial activity within affected dentin after minimally invasive treatment of caries lesions. Twenty-eight carious permanent teeth received standardized selective caries removal and random acid etch treatment (E) or not (NE) prior to adhesive restoration. Baseline and 3-month dentin biopsies were collected. The number of bacteria and activity of total bacterial cells and Streptococcus mutans were determined by quantitative PCR and RT-PCR. No statistically significant differences were observed in total bacterial number and activity between E and NE treatments (p > 0.3008). For NE, however, the residual S. mutans bacterial cells were reduced (p = 0.0027), while the activity per cell was significantly increased (p = 0.0010) after reentry at 3 months after restoration. This effect was not observed in group E. Although no significant differences were found between groups, this study suggests that acid etching of affected dentin prior to adhesive restoration may directly or indirectly have an inhibitive effect on the activity of residual cariogenic bacteria. Further research is required to investigate this potential effect. Copyright (C) 2010 S. Karger AG, Basel

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

Distribution of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in an Australian population

Background, aim: The present study describes (i) the natural distribution of the three putative periodontopathogens Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in an Australian population and (ii) the relationship between these organisms, pocket depths and supragingival plaque scores. Methods: Subgingival plaque was collected from the shallowest and deepest probing site in each sextant of the dentition. In total, 6030 subgingival plaque samples were collected from 504 subjects. An ELISA utilising pathogen-specific monoclonal antibodies was used to quantitate bacterial numbers. Results:: A. actinomycetemcomitans was the most frequently detected organism (22.8% of subjects) followed by P. gingivalis and P. intermedia (14.7% and 9.5% of subjects respectively). The majority of infected subjects (83%) were colonised by a single species of organism. A. actinomyceteincomitans presence was overrepresented in the youngest age group but under-represented in the older age groups. Conversely, P. gingivalis and P. intermedia presence was under-represented in the youngest age group but over-represented in the older age groups. Differing trends in the distribution of these bacteria were observed between subjects depending upon the site of the infection or whether a single or mixed infection was present; however, these differences did not reach significance. Bacterial presence was strongly associated with pocket depth for both A. actinomyceteincomitans and P. gingivalis. For A. actinomycetemcomitans, the odds of a site containing this bacterium decrease with deeper pockets. In contrast, for P. gingivalis the odds of a site being positive are almost six times greater for pockets >3 ram than for pockets less than or equal to3 nun. These odds increase further to 15.3 for pockets deeper than 5 mm. The odds of a site being P. intermedia positive were marginally greater (1.16) for pockets deeper than 3 mm. Conclusions: This cross-sectional study in a volunteer Australian population, demonstrated recognised periodontal pathogens occur as part of the flora of the subgingival plaque. Prospective longitudinal studies are needed to examine the positive relationship between pocket depth and pathogen presence with periodontal disease initiation and/or progression.

A microbiological study of Papillon-LeFevre syndrome in two patients

Aim-To analyse the microflora of subgingival plaque from patients with Papillon-Lefevre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction. Methods-Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. Results-The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S constellatus, S oralis, and S sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P melaninogenica, and P nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers. Conclusions-Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.

Acquisition and loss of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia over a 5-year period: effect of a triclosan/copolymer dentifrice

Objectives: The present study describes the natural history of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia over a 5-year period and the effect of a triclosan/copolymer dentifrice on these organisms in a normal adult population. Material and Methods: Subgingival plaque samples were collected from 504 adult volunteers. Probing pocket depths (PPD) and relative attachment levels were measured using an automated probe. Participants were matched for disease status (CPI), plaque index, age and gender, and allocated to receive either a triclosan/copolymer or placebo dentifrice. Re-examination and subgingival plaque sampling was repeated after 1, 2, 3, 4 and 5 years. P. gingivalis, A. actinomycetemcomitans and P. intermedia were detected and quantitated using an enzyme linked immunosorbent assay. Logistic regression and generalised linear modelling were used to analyse the data. Results: This 5-year longitudinal study showed considerable volatility in acquisition and loss (below the level of detection) of all three organisms in this population. Relatively few subjects had these organisms on multiple occasions. While P. gingivalis was related to loss of attachment and to PPD greater than or equal to3.5 mm, there was no relationship between A. actinomycetemcomitans or P. intermedia and disease progression over the 5 years of the study. Smokers with P. gingivalis had more PPD greater than or equal to3.5 mm than smokers without this organism. There was no significant effect of the triclosan dentifrice on P. gingivalis or A. actinomycetemcomitans . Subjects using triclosan were more likely to have P. intermedia than those not using the dentifrice; however this did not translate into these subjects having higher levels of P. intermedia and its presence was uniform showing no signs of increasing over the course of the study. Conclusion: The present 5-year longitudinal study has shown the transient nature of colonisation with P. gingivalis , A. actinomycetemcomitans and P. intermedia in a normal adult population. The use of a triclosan-containing dentifrice did not lead to an overgrowth of these organisms. The clinical effect of the dentifrice would appear to be independent of its antimicrobial properties.

Infectious agents in coronary atheromas: a possible role in the pathogenesis of plaque rupture and acute myocardial infarction

In this review we report our recent findings of histopathological features of plaque instability and the association with Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP) infection, studying thrombosed coronary artery segments (CAS) of patients who died due to acute myocardial infarction. Vulnerable plaques are known to be associated with fat atheromas and inflammation of the plaque. Here we demonstrated that vulnerability is also related with focal positive vessel remodeling that maintains relatively well preserved lumen even in the presence of large atheromatous plaques. This phenomena may explain why the cinecoronariography may not detect large and dangerous vulnerable plaques. Greater amount of these bacteria in vulnerable plaques is associated with adventitial inflammation and positive vessel remodeling: the mean numbers of lymphocytes were significantly higher in adventitia than in the plaque, good direct correlation was obtained between numbers of CD20 B cells and numbers of CP infected cells in adventitia, and between % area of MP-DNA in the plaque and cross sectional area of the vessel, suggesting a cause-effect relationship. Mycoplasma is a bacterium that needs cholesterol for proliferation and may increase virulence of other infectious agents. In conclusion, co-infection by Mycoplasma pneumoniae and Chlamydia pneumoniae may represent an important co-factor for plaque instability, leading to coronary plaque thrombosis and acute myocardial infarction, since larger amount of these bacteria strongly correlated with histological signs of more vulnerability of the plaque. The search of CMV and Helicobacter pilori in these tissues resulted negative.