The predominant cultivable dental plaque flora of beagle dogs with periodontitis

Abstract The predominant cultivable dental plaque flora was studied in 10 adult female beagle dogs with advanced periodontitis. Supragingival and subgingival plaque from a maxillary third premolar (P³) was removed and cultured anaerobically on various growth media and all colonies were subcultured and partially characterized. Histopathological specimens of the plaque sampling sites showed significant loss of connective tissue attachment. Spirochetes were found in ail samples. Anaerobic gram-negative organisms were predominant in both types of plaque accounting for about 55 % of the cultivable organisms in the supragingival plaque and almost 75% in the subgingival plaque. Bacteroides asaccharolyticus was the most prominant organism in the supragingival plaque, whereas Fusobacterium nucleatum predominated in the subgingival flora. Streptococcal and actinomycotic species were common in the supragingival plaque, but their proportions, especially those of the actmomycetes, were decreased in the subgingival flora. In many respects the bacterial profile associated with disease resembled that reported in human periodontal disease.     

The effect of intensive antibacterial therapy on the sulcular environment in monkeys. Part I. Changes in the bacteriology of the gingival sulcus

The changes induced in the bacteriology of the gingival sulcus were evaluated as part of a study considering the keratinizing potential of the sulcular epithelium when bacterial plaque was essentially eliminated. Two Rhesus monkeys were scaled and placed on a daily therapeutic regimen which included a prophylaxis, systemic tetracycline, and topical chlorhexidine. Over the 40 day experimental period and 74 days post-therapy, subgingival plaque samples were taken periodically. The plaque samples were cultured anaerobically and aerobically to determine the predominant bacterial flora. The total cultivable bacterial flora decreased from initial levels by greater than 99.9% with the antibacterial therapy. The flora shifted with therapy from one dominated by anaerobic organisms, including Bacteroides melaninogenicus (18%) and Fusobacterium species (13.9%), to a flora dominated by organisms growing aerobically. During treatment B. melaninogenicus and Fusobacterium species were not detected in any sample. After cessation of all therapy the anaerobes increased to dominance again, but B. melaninogenicus remained undetectable through 74 days post-therapy.     

The predominant cultivable dental plaque flora of beagle dogs with gingivitis

The predominant dental plaque flora of 15 female beagle dogs (1,3 and 6 year old) with naturally developed gingivitis was studied using a continuous anaerobic culturing technique. Supra- and subgingival plaque samples from the buccal aspect of the upper third premolar were cultured on various growth media and the organisms were partially characterized. The flora in all dogs was composed mostly of anaerobic Gram negative organisms. B. asaccharolyticus (B. melaninogenicus ss. asaccharolyticus) was found in both types of plaques in all animals and decomposed hydrogen peroxide suggesting catalase activity. F. nucleatum was found in higher proportions in the subgingival plaque as compared to B. asaccharolyticus and actinomycetes. Spirochetes were found in 10 of 15 supra- and in 1 of 15 subgingival specimens. S. mutans, S. mitior, S. salivarius, Veillonella, Selenomonas or vibrios could not be detected in any of the plaque samples. A combined data analysis showed, that the total viable CFU and the proportions of Gram positive organisms were significantly higher in the supra- than the subgingival plaque. Although the proportions of Gram negative bacteria were higher in subgingival plaque, the differences between the two types of plaques excluding F. nucleatum were not significant.     

Microbial Analysis of Subgingival Plaque Samples Compared to That of Whole Saliva in Patients With Periodontitis

Background: The detection of special bacterial species in patients with periodontitis is considered to be useful for clinical diagnosis and treatment. The collection of subgingival plaque samples is the common way for the determination of periodontopathic bacteria. However, recently, salivary analysis has been discussed as an advantageous future diagnostic method for periodontitis because it offers simple quantitative sampling and the possibility to assess various bacteria. The aim of this cross‐sectional study is to investigate whether there is a correlation between the results of different bacterial species in saliva and subgingival plaque samples from individuals with aggressive periodontitis (AgP) and chronic periodontitis (CP). Methods: Whole saliva and subgingival plaque samples from the deepest pocket of each quadrant were collected from 43 patients with CP and 33 patients with AgP. Twenty different bacterial species from both samplings were determined by the 16S ribosomal RNA‐based polymerase chain reaction with microarray technique. Results: All bacterial species were detected in salivary and subgingival plaque samples. For Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as well as Actinomyces viscosus, Campylobacter rectus/showae, Prevotella intermedia, Parvimonas micra, Eubacterium nodatum, and Campylobacter gracilis, a significant positive correlation between salivary and subgingival plaque samples was detected in patients with both types of periodontitis. There were no significant differences in bacteria in salivary and subgingival plaque samples between AgP and CP. Conclusion: Salivary analysis might be discussed as a potential alternative to subgingival plaque sampling for microbiologic analysis in both AgP and CP.     

Detection frequency of periodontitis‐associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤P<0.05). Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.     

Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis

Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years). Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA‐DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons. Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species. Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible rôle of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.     

The predominant cultivable subgingival flora of beagle dogs following ligature placement and metronidazole therapy

Ligature-induced periodontitis was evaluated microbiologically in 8 beagle dogs. Gingival health was established by repeated cleaning. At day 0 subgingival plaque was sampled from individual sites in two quadrants. All teeth in one quadrant were then ligated at the CEJ, and the other quadrant served as a non-ligated control which was cleaned three times each week. At day 14, four dogs were given metronidazole for seven days. Plaque was cultured anaerobically on nonselcctive media, and the predominant cultivable flora was characterized. At day 0 Gram-negative facultative rods represented 56.8 % of the flora. with motile and surface translocating organisms predominating. At day 14 Bacteroides asaccharolyticus , including catalase positive B.asaccharolyticus-like organisms, increased at ligated sites to 34.7 % of the cultivable flora. After metronidazole therapy the total bacterial count decreased, and Gram-negative anaerobic rods became non-detectable. Gram negative facultative bacteria which were motile or surface translocating represented 52.7 % of the cultivable flora after metronidazole treatment.
In the beagle dog ligature placement was associated with a shift in the flora from Gram-negative facultative rods to Gram-negative anaerobic rods. Metronidazole treatment reduced the total cultivable flora and selectively reversed the microflora shifts which followed ligature placement.     

Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤ P< 0.05). Two species ( Mogibacterium timidum and Porphyromonas gingivalis ) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects ( P< 0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.     

The effects of sustained release doxycycline on the anaerobic flora and antibiotic-resistant patterns in subgingival plaque and saliva

BACKGROUND: The purpose of this study was to determine the effects of periodontal treatment with a sustained-release, biodegradable gel containing 8.5% doxycycline on the anaerobic flora and on antibiotic susceptibility patterns associated with subgingival plaque and saliva. METHODS: Forty-five subjects with adult periodontitis were entered into a parallel design, single-blind study of 6 months' duration. The subjects were randomized to receive either doxycycline treatment (n = 23) or oral hygiene instruction/reinforcement (n = 22). Saliva and subgingival plaque samples were collected prior to and at 7, 21, 91, and 182 days after initiation of treatment. The proportion of the cultivable flora resistant to 10 microg doxycycline/ml was determined relative to total anaerobic counts, and the 3 most predominant colony types resistant to doxycycline were individually enumerated. A representative of each was subcultured, identified to genus and species level, and tested for its susceptibilities to 6 antibiotics. RESULTS: A significant decrease (P <0.01) in total anaerobic counts following doxycycline treatment caused a transient increase in the proportion, but not in the actual counts, of doxycycline-resistant bacteria recovered from both plaque and saliva at 7 and 21 days but not at 91 or 182 days. The same doxycycline-resistant taxa were recovered at all sample periods including baseline. Regardless of treatment, the isolates were similarly distributed and belonged to the same bacterial groups. CONCLUSIONS: Doxycycline treatment significantly reduced the anaerobic population in plaque but did not result in a change in either the number of resistant bacteria present or the acquisition of antibiotic resistance.     

The subgingival microbial flora during pregnancy

The subgingival bacterial flora from 2 gingival sites was cultured and characterized monthly in twenty periodontitis-free women during pregnancy and again post-partum. Monthly plaque samples were also cultured in eleven age and disease matched non-pregnant women. Plaque was processed anaerobically on selective and nonselective media and the predominant colony types were characterized. A portion of each plaque sample was tested for bacterial uptake of C¹⁴-estradiol and C¹⁴-progesterone. Plasma levels of estrogens and progesterone were measured four times in each subject. The number of gingival bleeding sites, the Gingival Index and the Plaque Index were determined at each sampling period.
In the second trimester there was a significant increase in gingivitis, the ratio of anaerobic to aerobic bacteria, and the proportional levels of Bacteroides melaninogenicus ss. intermedius. In the third trimester both gingivitis and the levels of B. melaninogenicus ss. intermedius decreased. Plaque uptake of C¹⁴-steroids increased significantly during pregnancy and paralleled the plaque levels of B. melaninogenicus ss. intermedius. In the second trimester, recovery of B. melaninogenicus ss. intermedius was strongly correlated with plasma levels of estrogens and progesterone. No changes were observed in clinical parameters or the subgingival flora of non-pregnant subjects.
Pregnancy and specifically steroid hormones appear capable of influencing the normal bacterial flora and inducing alterations in the subgingival ecology.     

Perpetuation of subgingival biofilms in an in vitro model

This study evaluated the reproducibility of in‐vitro‐grown biofilms, initiated with subgingival plaque from patients with periodontal disease, and continued through several cycles by re‐inoculating new biofilms from previously grown biofilms. Subgingival plaque samples from bleeding pockets along with saliva samples were collected from three patients with chronic periodontitis and perpetuated through seven cycles. Calcium hydroxyapatite disks were coated with sterilized saliva inoculated with dispersed subgingival plaque. The biofilms were grown anaerobically at 37°C for 10 days, and at specific intervals total viable bacteria were enumerated and the species present were analysed by DNA–DNA checkerboard hybridization. All cycles of biofilm growth occurred at similar rates and reached steady‐state at day 7. No statistically or microbially significant differences were found for viable counts or species present, at the same period of maturation, among the different cycles. This study demonstrated that growth of certain target subgingival periodontal species in this biofilm model was reproducible and could be perpetuated in vitro through several cycles. The model could be useful in future studies to characterize different periodontopathogenic properties and biofilm interactions, especially in recolonization studies.     

The effect of dietary Gantrisin® supplements on the flora of periodontal pockets in four beagle dogs

The predominant cultivable flora from advanced periodontal pockets (> 4 mm bone loss) was compared with the microbiota isolated from areas of incipient periodontal lesions (< 2 mm bone loss) in Beagle, dogs with periodontal disease. In addition the effect of long term dietary antibiotic (sulfisoxazole) administration on the microbial flora and the clinical course of periodontal disease in these animals was determined.
Four male Beagle dogs, 40 months old, had been maintained on a soft diet for 36 months prior to the time of bacterial sampling; two of the animals had been given 44 mg/Kg Gantrisin® (sulfisoxazole) p.o . daily for 3 years. Clinical and radiographic examination of the dogs demonstrated that Gantrisin® seemed to have no effect on the development of periodontitis since both groups exhibited clinical signs of periodontal disease and demonstrated a range of alveolar bone destruction from minimal to severe.
Bacterial samples were obtained and cultured from the base of each site utilizing anaerobic techniques. In addition, direct microscopy of whole plaque was carried out.
The distribution of the predominant cultivable flora in both treated and non-treated animals revealed that Gram positive organisms predominated in the incipient lesions, while Gram negative anaerobic rods predominated in advanced periodontal lesions. In all sites anaerobes outnumbered facultatively anaerobic bacteria by at least 2:1.     

Evaluation of the relationship between smoking during pregnancy and subgingival microbiota

BACKGROUND: Numerous studies have shown that smoking negatively affects periodontal health. Hormonal changes, which occur during pregnancy have also been reported to have adverse effects on the periodontal tissues or indirectly through alterations in the subgingival bacterial flora. At present, no knowledge exists concerning possible effects of smoking on the composition of subgingival plaque in pregnancy. The purpose of the present study was to evaluate the effects of smoking during pregnancy on the subgingival plaque bacteria most commonly associated with periodontal disease. METHODS: A total number of 181 women were examined within 72 h post-partum. Smoking status was recorded by means of a self-reported questionnaire and the study population was divided into three groups; non-smokers, light smokers, and heavy smokers. In each woman, two subgingival plaque samples were obtained from mesio- or disto-buccal aspect of randomly selected one molar and one incisor tooth by sterile paperpoints. Clinical periodontal recordings comprising presence of dental plaque, bleeding on probing (BOP), and probing pocket depth (PPD) were performed at six sites per each tooth at all teeth. Plaque samples were analysed by checkerboard DNA-DNA hybridization with respect to 12 bacterial species. In all analyses, the individual subject was the computational unit. Thus, mean values for all clinical parameters were calculated and bacterial scores from each individual sample were averaged. Statistical methods included chi2 test, Kruskal-Wallis test and Mann-Whitney U-test. RESULTS: Mean ages were similar in the study groups. Plaque, BOP and PPD recordings were lower in the heavy-smoker group, but the differences were not statistically significant (p>0.05). The detection rates and bacterial loads of the specific subgingival bacteria exhibited no significant differences between the groups. No correlation could be found between smoking status and detection rates and bacterial loads of various bacterial species. CONCLUSION: The present findings suggest that smoking during pregnancy does not have a significant effect on the composition of subgingival plaque bacteria.     

Evaluation of a non-radioactive DMA probe for detecting Porphyromonas gingivalis in subgingival specimens

This study compared the ability of a nonradioactive digoxigenin-labeled DNA probe and anaerobic culture to identify subgingival Porphyromonas gingivalis. Total cellular DNA from P. gingivalis ATCC 33277T was labeled using the Genius kit from Boehringer Mannheim Biochemicals. Anaerobic culture was performed using VMGA III transport medium and enriched brucella blood agar. The DNA probe could detect as little as 1000 P. gingivalis cells added to supragingival plaque. Also, the probe could detect P. gingivalis when it was present in proportions too low to be visualized on overgrown bacterial plates. The probe showed no visible reaction with strains of various oral species or with thousands of non-P. gingivalis colonies from plaque samples. VMGA III could maintain the viability of P. gingivalis for up to 6 days, as evidenced by DNA probing of colony blot of subgingival cultures. A total cellular DNA probe for detecting P. gingivalis seems to offer a simple and reliable method of detecting the organism in subgingival specimens.     

Multiple displacement amplification as an aid in checkerboard DNA–DNA hybridization

Objective: The study aimed to determine if multiple displacement amplification could be used to provide abundant target DNA and DNA probes for checkerboard DNA–DNA hybridization. Methods: Multiple displacement amplification was used to amplify 1 and 10 ng DNA from 16 individual bacterial species, DNA from single colonies, from a mixture of 20 bacterial species and oral biofilm samples, such as supragingival plaque, subgingival plaque, buccal swab and root canal samples. Samples in reaction buffer were heat‐denatured at 95°C for 3 min and cooled to 4°C. Φ29 DNA polymerase was added and the mixture was incubated at 30°C for 16–18 h. The quantity of the product was evaluated by the Picogreen assay. The amplified material was labeled with digoxigenin. The probes were compared with probes obtained from unamplified DNA using checkerboard DNA–DNA hybridization. Both amplified DNA and unamplified DNA were used as targets on the membrane. Amplified oral biofilm samples were compared to unamplified samples using checkerboard DNA–DNA hybridization. Results: The DNA yield ranged from 4 to 11 μg. DNA–DNA hybridization showed that the amplified genome of each species used either as target or as probe provided signals equivalent to controls and that amplification of a mixture of species provided signals comparable to those provided by the unamplified source mixture. Amplified oral biofilm samples exhibited comparable proportions of bacterial DNA when compared to the original unamplified samples. Conclusions: The multiple displacement amplification technique is a simple and reliable method to uniformly amplify DNA for use in checkerboard DNA–DNA hybridization. It is also a useful tool in the amplification of clinical samples.     

Early microbial succession in redeveloping dental biofilms in periodontal health and disease

Teles FR, Teles RP, Uzel NG, Song XQ, Torresyap G, Socransky SS, Haffajee AD. Early microbial succession in redeveloping dental biofilms in periodontal health and disease. J Periodont Res 2012; 47: 95–104. © 2011 John Wiley & Sons A/S Background and Objective: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. Material and Methods: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA–DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time‐point. Ecological succession was determined using a modified moving‐window analysis. Results: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 d. At 4–7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. Conclusion: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.     

“Reverse” DMA hybridization method for the rapid identification of subgingival microorganisms

A “reverse” hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of “unknown” strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples     

The subgingival microbiota of Papillon-Lefèvre syndrome

Background: There is little information about the microbiologic profiles of periodontal lesions in Papillon‐Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA‐based Human Oral Microbe Identification Microarray (HOMIM). Methods. Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. Results: The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high‐frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. Conclusions: The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.     

An in vitro biofilm model of subgingival plaque

INTRODUCTION: Numerous biofilm models have been described for the study of bacteria associated with the supragingival plaque. However, there are fewer models available for the study of subgingival plaque. The purpose of this study was to develop and validate a model that closely mimicked the composition of the subgingival flora. METHODS: The model was developed as follows: calcium hydroxyapatite disks were coated overnight with 10% sterile saliva, placed in flat-bottomed tissue culture plates containing trypticase-soy broth, directly inoculated with a small aliquot of dispersed subgingival plaque, incubated anaerobically, and transferred to fresh medium at 48-h intervals until climax (steady-state) biofilms were formed ( approximately 10 days). RESULTS: The model, based on samples from eight periodontitis patients and eight healthy subjects, yielded a multi-species, heterogeneous biofilm, consisting of both gram-positive and gram-negative species, and comprising 15-20 cultivable species associated with the subgingival flora. The species present and their proportions were reflective of the initial cultivable subgingival flora. Comparisons of the initial plaque samples from healthy subjects and the mature biofilms showed 81% similarity in species and 70% similarity in the proportions present. Biofilms formed from samples obtained from periodontally diseased subjects were 69% similar in species and 57% similar in the proportions present. CONCLUSIONS: The biofilm model described here closely reproduces the composition of the cultivable subgingival plaque both in the species present and in their relative proportions. Differences existed between biofilms grown from diseased and non-diseased sites with the former being characterized by the presence of periodontal pathogens at microbially significant levels.     

Supragingival and subgingival microbiota of adult patients with Down’s syndrome. Changes after periodontal treatment

In this longitudinal study, five adult Down’s syndrome patients with periodontitis were placed on a frequent recall visit schedule (every 6 weeks) after treatment, in order to investigate: 1) the microbiological status, both supragingivally and subgingivally, and the changes that occurred after treatment and 2) the effect of frequent professional supragingival plaque control on the subgingival microbiota and clinical variables in these patients. The clinical variables recorded were probing pocket depth, probing attachment level, bleeding on probing and presence of plaque (full mouth, six surfaces per tooth). Microbiological examination was performed separately for supragingival and subgingival samples from the same site for 14 species, using whole genomic DNA probes and the “checkerboard” DNA‐DNA hybridization technique. The findings indicate that, although a reduction of periodontal indices was noticed, plaque levels remained high (60%) even at the end of the experimental period. Periodontal pathogens including Porphyromonas gingivalis, Bacteroides forsythus and Actinobacillus actinomycetemcomitans were frequently detected both supragingivally and subgingivally (>30%). The presence of a species supragingivally and the presence at the same time points subgingivally were correlated. This finding suggested that supragingival plaque acts as a reservoir for reinfection of treated sites. A reduction of the percentages of detection of these species was noticed 1 month after an oral hygiene period as well as at 3 and 6 months after treatment. Inadequate oral hygiene as performed by these patients probably affected supragingival, and consequently subgingival, plaque composition.