Formation of biofilm on various implant abutment materials.

The characteristics of prosthetic implant components, such as the type, material, and surface roughness of abutments, can affect biofilm formation. Since an ideal abutment surface for the reduction of bacterial adhesion has yet to be found, this in vitro study aimed to quantify biofilm formation on laser-treated titanium, zirconia, and titanium surfaces. Sterile titanium, zirconia, and laser-treated titanium discs were placed in sterile 48-well plates. Biofilm formation was induced by adding sterilized, unstimulated human saliva and suspensions of Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), and Prevotella intermedia (Pi) to the wells. Viable bacteria in the biofilm were quantified with real-time polymerase chain reaction in conjunction with propidium monoazide. The disc material, the type of bacteria, and their interactions had significant effects on the bacterial counts. On all surfaces, the Pg count was significantly higher than both the Pi and Aa counts (P = 0.0001). The highest count of periodontal pathogens was found on laser-treated surfaces. The second highest and the lowest counts were found on zirconia and titanium surfaces, respectively.

In Vitro Effects of Polyphosphate against Prevotella intermedia in Planktonic Phase and Biofilm.

Polyphosphate (polyP) has gained a wide interest in the food industry due to its potential as a decontaminating agent. In this study, we examined the effect of sodium tripolyphosphate (polyP3; Na5P3O10) against planktonic and biofilm cells of Prevotella intermedia, a major oral pathogen. The MIC of polyP3 against P. intermedia ATCC 49046 determined by agar dilution method was 0.075%, while 0.05% polyP3 was bactericidal against P. intermedia in time-kill analysis performed using liquid medium. A crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that sub-MICs of polyP3 significantly decreased biofilm formation. Under the scanning electron microscope, decreased numbers of P. intermedia cells forming the biofilms were observed when the bacterial cells were incubated with 0.025% or higher concentrations of polyP3. Assessment of biofilm viability with LIVE/DEAD staining and viable cell count methods showed that 0.05% or higher concentrations of polyP3 significantly decreased the viability of the preformed biofilms in a concentration-dependent manner. The zone sizes of alpha-hemolysis formed on horse blood agar produced by P. intermedia were decreased in the presence of polyP3. The expression of the genes encoding hemolysins and the genes of the hemin uptake (hmu) locus was downregulated by polyP3. Collectively, our results show that polyP is an effective antimicrobial agent against P. intermedia in biofilms as well as planktonic phase, interfering with the process of hemin acquisition by the bacterium.

In Vitro Evaluation of Planktonic Growth on Experimental Cement-Retained Titanium Surfaces.

BACKGROUND The purpose of this study was to compare the effects of selected cements, or their combination with titanium, on the growth of two periodontopathic bacteria: Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn). MATERIAL AND METHODS This study was comprised of several experimental groups: 1) Dental luting cements (glass ionomer cement, methacrylate-based resin cement, zinc-oxide eugenol cement, eugenol-free zinc oxide cement; 2) titanium discs; and 3) titanium combination cement discs. The disks were submerged in bacterial suspensions of either Fn or Pi. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600). Mean and standard deviations were calculated for planktonic growth from three separate experiments. RESULTS Intergroup comparison of all experimental groups revealed increased growth of Pi associated with cement-titanium specimens in comparison with cement specimens. Regarding the comparison of all groups for Fn, there was an increased amount of bacterial growth in cement-titanium specimens although the increase was not statistically significant. CONCLUSIONS The combination of cement with titanium may exacerbate the bacterial growth capacity of Pi and Fn in contrast to their sole effect.

Quantitation of biofilm and planktonic life forms of coexisting periodontal species.

BACKGROUND: Complexity of oral polymicrobial communities has prompted a need for developing in vitro models to study behavior of coexisting bacteria. Little knowledge is available of in vitro co-growth of several periodontitis-associated species without early colonizers of dental plaque. THE AIM: was to determine temporal changes in the quantities of six periodontal species in an in vitro biofilm model in comparison with parallel planktonic cultures. MATERIAL AND METHODS: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Parvimonas micra, Campylobacter rectus and Fusobacterium nucleatum were anaerobically grown as multispecies and monospecies biofilms and parallel planktonic cultures using cell culture plates and microfuge tubes, respectively. After incubating 2, 4, 6, 8 days, biofilms and planktonic cultures were harvested, DNA extracted and the target species quantified using qPCR with species-specific 16S rDNA primers. Biofilm growth as monocultures was visualized at day 2 and 8 with confocal microscopy and crystal violet staining. RESULTS: The six species were found throughout the test period in all culture conditions, except that P. gingivalis and F. nucleatum were not detected in multispecies planktonic cultures at day 8. In multispecies biofilm, P. gingivalis qPCR counts (cells/ml) increased (P<0.05) from day 2-8 and were then higher (P<0.05) than those of A. actinomycetemcomitans and C. rectus, whereas in monospecies biofilm, P. gingivalis counts were lower (P<0.05) than those of the other species, except A. actinomycetemcomitans. When multi- and monospecies biofilm cultures were compared, P. gingivalis counts were higher (P<0.05) but those of the other species, except P. intermedia, lower (P<0.05) in multispecies biofilm. Comparison between planktonic and biofilm cultures showed that A. actinomycetemcomitans, P. micra and C. rectus had higher (P<0.05) counts in planktonic cultures no matter whether grown in mono- or multispecies environment. CONCLUSIONS: Six periodontal species were able to form multispecies biofilm up to 8 days in vitro without pioneer plaque bacteria. P. gingivalis seemed to prefer multispecies biofilm environment whereas P. micra and A. actinomycetemcomitans planktonic culture.

Protective effect of an egg yolk-derived immunoglobulin (IgY) against Prevotella intermedia-mediated gingivitis.

AIMS: To investigate the effects of an egg yolk-derived immunoglobulin (IgY) specific to Prevotella intermedia in vitro and in vivo. METHODS AND RESULTS: An IgY specific to P. intermedia was produced by immunizing hens with formaldehyde-inactivated P. intermedia and showed high titres when subjected to an ELISA. The obtained IgY inhibited the growth of P. intermedia in a dose-dependent manner at concentrations from 1 to 20 mg ml(-1) in Center for Disease Control and Prevention liquid medium. Forty rats were challenged with P. intermedia on gingivae and then randomly divided into four groups, which were syringed respectively with phosphate-buffered saline, 1 mg ml(-1) of tinidazole, 20 mg ml(-1) of nonspecific IgY and 20 mg ml(-1) of the IgY specific to P. intermedia at a dosage of 300 µl per day. Gingival index (GI), plaque index (PI), bleeding on probing (BOP), counts of white blood cell (WBC) and histopathological slide of the gums were measured after treatment for 15 days. The gingivitis rats treated with the IgY specific to P. intermedia showed significantly decreased GI, PI, BOP and WBC (P < 0·05). Gum histopathology of the treated rats demonstrated a superior protective effect of the specific IgY on P. intermedia-mediated gingivitis. CONCLUSIONS: A new immunoglobulin specific to P. intermedia was developed from egg yolk. This specific IgY can dose-dependently inhibit the growth of P. intermedia and protect rats from gingivitis induced by P. intermedia. SIGNIFICANCE AND IMPACT OF THE STUDY: The new IgY has potential for the treatment of P. intermedia-mediated gingivitis.

Validation of a quantitative real-time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species-specific quantification of an in vitro subgingival biofilm model.

BACKGROUND AND OBJECTIVE: Subgingival biofilms are the prime etiological factor of periodontal disease. Owing to their complex polymicrobial nature, quantification of individual bacterial species within the biofilm for research and diagnostic purposes can be methodologically challenging. The aims of this study were to establish a quantitative real-time PCR (qPCR) assay to quantify the bacteria used in our 10-species in vitro 'subgingival' biofilm model and to compare the quantitative outcome with fluorescence microscopy and colony-forming unit (CFU) counts on selective agar plates. MATERIAL AND METHODS: The 10 species included in the in vitro biofilm were Streptococcus oralis, Streptococcus anginosus, Veillonella dispar, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Actinomyces oris, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia. The numbers of each species were quantified at two time points using qPCR, microscopy counting following fluorescence in-situ hybridization (FISH) or immunofluorescence staining, and counting of CFUs after growth on selective agar plates. RESULTS: All 10 species were successfully quantified using qPCR and FISH or immunofluorescence, and the eight species culturable on selective agar plates were also quantified by counting the numbers of CFUs after growth on selective agar. In early biofilm cultures, all methods showed a significant correlation, although the absolute numbers differed between methods. In late biofilm cultures, measurements obtained using qPCR and FISH or immunofluorescence, but not by CFU counts, maintained significant correlation. CFU counts yielded lower values than did measurements made using the other two methods. CONCLUSION: Quantitative PCR and epifluorescence microscopy can be easily combined with each other to determine species-specific bacterial numbers within biofilms. However, conventional bacterial cultures cannot be as efficiently combined using these molecular detection methods. This may be crucial in designing and selecting appropriate clinical diagnostic methods for subgingival biofilm samples.

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers.

OBJECTIVE: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. MATERIALS AND METHODS: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher's exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. RESULTS: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue-coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. CONCLUSION: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.

Early bacterial colonization and soft tissue health around zirconia and titanium abutments: an in vivo study in man.

AIM: To compare the early bacterial colonization and soft tissue health of mucosa adjacent to zirconia (ZrO(2)) and titanium (Ti) abutment surfaces in vivo. MATERIALS AND METHODS: Twenty edentulous subjects received two endosseous mandibular implants. The implants were fitted with either a ZrO(2) or a Ti abutment (non-submerged implant placement, within-subject comparison, left-right randomization). Sulcular bacterial sampling and the assessment of probing pocket depth, recession and bleeding on probing were performed at 2 weeks and 3 months post-surgery. Wilcoxon matched-pairs, sign-rank tests were applied to test differences in the counts of seven marker bacteria and the clinical parameters that were associated with the ZrO(2) and Ti abutments, at the two observation time points. RESULTS: ZrO(2) and Ti abutments harboured similar counts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Peptostreptococcus micros, Fusobacterium nucleatum and Treponema denticola at 2 weeks and 3 months. Healthy clinical conditions were seen around both ZrO(2) and Ti abutments at all times, without significant differences in most clinical parameters of peri-implant soft tissue health. Mean probing depths around Ti abutments were slightly deeper than around ZrO(2) abutments after 3 months (2.2 SD 0.8 mm vs. 1.7 SD 0.7 mm, P=0.03). CONCLUSIONS: No difference in health of the soft tissues adjacent to ZrO(2) and Ti abutment surfaces or in early bacterial colonization could be demonstrated, although somewhat shallower probing depths were observed around ZrO(2) abutments after 3 month.

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis.

BACKGROUND: Patients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of CF lung disease is unknown. METHODS: The virulence of Prevotella intermedia and Ps aeruginosa was investigated in vitro and in mice, antibodies against P intermedia in CF sera were assessed and a culture-independent detection method for P intermedia/P nigrescens in CF sputum was tested. RESULTS: P intermedia reached cell numbers of >10(5)->10(7) colony-forming units (CFU)/ml sputum. The majority of patients with CF (16/17; 94.1%) produced antibodies against two immunoreactive antigens of P intermedia. Culture supernatant fluids, collected from 10(9) P intermedia cells, were more cytotoxic to respiratory epithelial cells in vitro and inflammatory in mouse lungs than respective fluids from anaerobically grown Ps aeruginosa, while fluids from aerobically grown Ps aeruginosa had the highest cytotoxicity and inflammation. Both pathological effects were largely reduced when culture supernatant fluids from 10(7) cells of either species were used. P intermedia cells (∼10(6)CFU/lung) did not induce mortality in the agar beads lung infection mouse model, while Ps aeruginosa cells caused death in 30% of mice due to rapid multiplication. A P intermedia/P nigrescens-specific PNA probe was significantly more sensitive than culture-dependent diagnostic assays to detect these strict anaerobes. CONCLUSION: Ps aeruginosa and P intermedia become significantly virulent in vitro and in vivo when cell numbers exceed 10(8) CFU/lung.

Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis and Prevotella intermedia.

Lactoferrin (LF) is an iron-binding antimicrobial protein present in saliva and gingival crevicular fluids, and it is possibly associated with host defense against oral pathogens, including periodontopathic bacteria. In the present study, we evaluated the in vitro effects of LF-related agents on the growth and biofilm formation of two periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, which reside as biofilms in the subgingival plaque. The planktonic growth of P. gingivalis and P. intermedia was suppressed for up to 5 h by incubation with >or=130 microg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and >or=6 microg/ml of bLF-derived antimicrobial peptide lactoferricin B (LFcin B); but those effects were weak after 8 h. The biofilm formation of P. gingivalis and P. intermedia over 24 h was effectively inhibited by lower concentrations (>or=8 microg/ml) of various iron-bound forms (the apo, native, and holo forms) of bLF and hLF but not LFcin B. A preformed biofilm of P. gingivalis and P. intermedia was also reduced by incubation with various iron-bound bLFs, hLF, and LFcin B for 5 h. In an examination of the effectiveness of native bLF when it was used in combination with four antibiotics, it was found that treatment with ciprofloxacin, clarithromycin, and minocycline in combination with native bLF for 24 h reduced the amount of a preformed biofilm of P. gingivalis compared with the level of reduction achieved with each agent alone. These results demonstrate the antibiofilm activity of LF with lower iron dependency against P. gingivalis and P. intermedia and the potential usefulness of LF for the prevention and treatment of periodontal diseases and as adjunct therapy for periodontal diseases.

Does the frequency of Prevotella intermedia increase during pregnancy?

INTRODUCTION: The former Bacteroides intermedius, currently including Prevotella intermedia and Prevotella nigrescens, has been associated with hormone-induced pregnancy gingivitis. The aim of the present longitudinal study was to determine whether only P. intermedia or P. nigrescens, or both species, are involved in the demonstrated microbial shift during pregnancy. METHODS: Subgingival plaque and saliva samples, collected from 30 healthy pregnant women and 24 healthy non-pregnant women as their controls, were examined for the presence of pigmented gram-negative anaerobes. Altogether 2628 isolates were preliminarily identified as P. intermedia sensu lato, based on phenotypic testing. Their further identification was performed by using a 16S ribosomal DNA-based polymerase chain reaction (PCR). RESULTS: A mean of 8.3 P. intermedia sensu lato isolates from each subject/sampling was examined. During the second trimester, the mean number of P. intermedia sensu lato in plaque increased along with increasing signs of pregnancy gingivitis, and then both decreased. After delivery, gingival inflammation still decreased while the number of P. intermedia sensu lato transiently increased both in plaque and saliva. In the present study, the vast majority of isolates (95.3%) proved to be P. nigrescens and 2.5% were P. intermedia. The remaining 2.2% of the isolates could not be identified with PCR as P. intermedia or P. nigrescens. The corresponding percentages in the control population were 94.2%, 5.5%, and 0.3%. CONCLUSION: In the oral cavity of relatively young women without periodontitis, P. nigrescens, unlike P. intermedia, is a frequent finding. Conceivably, pregnant women harbor increasing numbers of P. nigrescens associated with pregnancy gingivitis.

Identification of Periopathogenes from Dental Plaque in Periodontal Patients with PCR Technique and Their Association with Composite Interleukin-1 Genotype.

INTRODUCTION: The present study aimed to assess the presence of main types of microorganisms involved in the aetiopathogenesis of chronic periodontitis with PCR technique and determinates the presence of composite IL-1 genotype and their associations with founded bacteria. MATERIAL AND METHOD: The examined group was consisted from 20 subjects with diagnosed chronic periodontitis and 20 healthy control without periodontitis. Clinical parameters like gingival index (GI), plaque index (PI), bleeding on probing (BOP), periodontal pocket depth (PPD) and clinical attachment lost (CAL) were determinates. Subgingival dental plaque was collected using a sterilized paper point. We used Parodontose Plus test, reverse hybridization kit, for the detection of periodontal marker bacteria, as well as for the detection of composite Interleukin -1 Genotype Results: The most present bacterial species detected from subgingival dental plaque was Treponema denticola and Porfiromonas gingivalis which was present in 65% of examined patients. In relation to the presence of positive genotype in patients, there was no significant difference between the test and control group for p> 0.05 (p = 1.00). For χ2=8,17 (p=0,06, p<0,05) there is an association between Prevotella intermedia, and composite genotype. Between positive genotype and analyzed bacterial species A. actinomycetem comitans for p> 0.05 (p = 1.00), P. gingivalis for p> 0.05 (p = 0.16), T. Forsythia for p> 0.05 (p = 0.20), T. Denticola for p> 0.05 (p = 0.64) no association was found. CONCLUSION: This investigations confirmed the strong association of these five examined periopathogenes with periodontitis.

Effects of Lactobacillus salivarius WB21 combined with green tea catechins on dental caries, periodontitis, and oral malodor.

OBJECTIVE: To evaluate the combined use of Lactobacillus salivarius WB21 and (-)-epigallocatechin gallate (EGCg) for oral health maintenance. DESIGN: The effects of L. salivarius WB21 on growth of Streptococcus mutans, the insoluble glucan produced by S. mutans, and on growth of Porphyromonas gingivalis were evaluated in vitro. In addition, the susceptibility of five oral pathogenic bacteria and L. salivarius WB21 to EGCg, the inhibiting effect of EGCg on methyl mercaptan, and the effects of L. salivarius WB21 and EGCg in combination on growth of P. gingivalis were examined. RESULTS: Lactobacillus salivarius WB21 showed concentration-dependent inhibition of the growth of S. mutans. Addition of L. salivarius WB21 inhibited production of the insoluble glucan by S. mutans (p < 0.001). A filtrate of L. salivarius WB21 culture solution inhibited growth of P. gingivalis (p < 0.001 vs. control), and this effect was enhanced when it was used in combination with EGCg (p < 0.001 vs. the addition of L. salivarius WB21). In addition, EGCg directly inhibited methyl mercaptan in a concentration-dependent manner (p < 0.001). Concerning bacterial susceptibility to EGCg, growth of P. gingivalis, Prevotella intermedia, and Fusobacterium nucleatum was inhibited at 2.5 mg/mL of EGCg, while that of L. salivarius WB21 was inhibited at 25 mg/mL EGCg. CONCLUSIONS: Our results imply that L. salivarius WB21 may be useful for controlling dental caries, periodontitis, and oral malodor. In addition, the effects of L. salivarius WB21 on periodontitis and oral malodor may be synergistically enhanced by use in combination with EGCg.

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria.

INTRODUCTION: During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. METHODS: GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and alpha-defensins (HNP1-3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. RESULTS: Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1-3 in samples from patients, compared to controls. The amount of HNP1-3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. CONCLUSION: The lack of a correlation between LL-37, HNP1-3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.

Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis.

BACKGROUND: The purpose of this study was to test the hypothesis that periodontal pathogens associated with aggressive periodontitis persist in extracrevicular locations following scaling and root planing, systemic antibiotics, and antimicrobial rinses. METHODS: Eighteen patients with aggressive periodontitis received a clinical examination during which samples of subgingival plaque and buccal epithelial cells were obtained. Treatment consisted of full-mouth root planing, systemic antibiotics, and chlorhexidine rinses. Clinical measurements and sampling were repeated at 3 and 6 months. Quantitative polymerase chain reaction determined the number of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), and Treponema denticola in the plaque. Fluorescence in situ hybridization and confocal microscopy determined the extent of intracellular invasion in epithelial cells. RESULTS: Clinical measurements improved significantly following treatment. All bacterial species except P. gingivalis were significantly reduced in plaque between baseline and 3 months. However, all species showed a trend to repopulate between 3 and 6 months. This increase was statistically significant for log T. denticola counts. All species were detected intracellularly. The percentage of cells infected intracellularly was not affected by therapy. CONCLUSIONS: The 6-month increasing trend in the levels of plaque bacteria suggests that subgingival recolonization was occurring. Because the presence of these species within epithelial cells was not altered after treatment, it is plausible that recolonization may occur from the oral mucosa. Systemic antibiotics and topical chlorhexidine did not reduce the percentage of invaded epithelial cells. These data support the hypothesis that extracrevicular reservoirs of bacteria exist, which might contribute to recurrent or refractory disease in some patients.

Periodontopathogenic bacteria multiply in the environmental amoeba Acanthamoeba castellani.

Porphyromonas gingivalis and Prevotella intermedia are obligate anaerobe gram-negative periodontopathogenic bacteria. Free-living amoebae, such as Acanthamoeba spp., are well known as environmental hosts of several human pathogens, such as Franciscella tularensis, Chlamydia pneumoniae, Legionella pneumophila and Mycobacteria spp. This study tested the ability of P. gingivalis and P. intermedia to become internalized, to survive and replicate in Acanthamoeba castellani. Our results show for the first time that P. gingivalis and P. intermedia isolated from periodontitis patients are capable of infecting A. castellani cells in vitro and are able to survive and multiply intracellularly. From our experimental data it can be suggested that periodontopathogenic bacteria might be conditioned in the evolution by amoebae and free-living amoebae can act as an environmental reservoir for these pathogens.

Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals.

OBJECTIVES: To measure antibacterial action of photoactivated disinfection (PAD) on endodontic bacteria in planktonic suspension and root canals. METHODS: Four bacteria, Fusobacterium nucleatum,Peptostreptococcus micros, Prevotella intermedia and Streptococcus intermedius, were tested in suspension. After mixing equal volumes of Tolonium chloride and bacterial suspension for 60s, each 200 microL of concentration (>10(6)cfu mL(-1)) was irradiated with light at 633+/-2 nm. Each energy dose/Tolonium chloride concentration combination was tested eight times, with controls. Prepared root canals in Training Blocs and extracted human teeth were inoculated with S. intermedius followed by 10 mg L(-1) Tolonium chloride or saline. Bacteria in canals were sampled before and after light irradiation. Student t-test assessed significance of changes in viable bacteria produced by treatment of either light or Tolonium chloride alone and light/Tolonium chloride combinations. RESULTS: In suspension, reductions in bacteria were highly significant (P<0.01) for light/Tolonium chloride combinations compared to light or Tolonium chloride alone. Maximum mean log reductions of 1.14 (P. intermedia), 2.48 (P. micros), 2.81 (F. nucleatum) and 6.73 (S. intermedius) were at 4.8 J/20 mg L(-1). Antibacterial action was increased by energy dose increase (not always significantly), but not by Tolonium chloride concentration. In control canals mean log reductions of 0.42 (Blocs) and 0.38 (teeth) from initial levels were not significant. PAD mean log reductions of 2.40 (Blocs) and 2.01 (teeth) were highly significant. Changes for PAD/energy dose combinations were not significant. CONCLUSION: PAD killed endodontic bacteria at statistically significant levels compared to controls. Kills varied with bacterial species.

A Preliminary Study of the Effects of pH upon Fluorescence in Suspensions of Prevotella intermedia.

The quantification of fluorescence in dental plaque is currently being developed as a diagnostic tool to help inform and improve oral health. The oral anaerobe Prevotella intermedia exhibits red fluorescence due to the accumulation of porphyrins. pH affects the fluorescence of abiotic preparations of porphyrins caused by changes in speciation between monomers, higher aggregates and dimers, but this phenomenon has not been demonstrated in bacteria. Fluorescence spectra were obtained from suspensions of P. intermedia that were adjusted to pHs commensurate with the range found within dental plaque. Two fluorescent motifs were identified; 410 nm excitation / 634 nm emission (peak A) and 398 nm excitation / 622 nm emission (peak B). A transition in the fluorescence spectra was observed from peak A to peak B with increasing pH which was also evident as culture age increased from 24 hours to 96 hours. In addition to these 'blue-shifts', the intensity of peak A increased with pH whilst decreasing with culture age from 24 to 96 hours. A bacterium's relationship with the local physiochemical environment at the time of image capture may therefore affect the quantification of dental plaque fluorescence.

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.