Rediscovering Sig Socransky, the genius and his legacy.

Some individuals make contributions so vital to their field of knowledge that their names become almost synonymous with that field. This is the case of Sig Socransky and the field of periodontal microbiology. Sig Socransky, or simply Sig, was born in Toronto, Canada and received his DDS degree from the University of Toronto in 1957. He studied microbiology and periodontology at Harvard, receiving a certificate in 1961. That same year he was recruited to work as a Research Associate at the Forsyth Dental Center. In 1968, he was nominated Senior Member of the Staff and Head of the Department of Periodontology. During his 50-year career at Forsyth, Sig published over 300 manuscripts, keeping an average of 7 publications per year. His work had an indelible impact in the fields of periodontology and oral microbiology. All these accomplishments pale in comparison with the impact that Sig had on a personal level. We have collected testimonials from some of his former students, closest collaborators, and friends in an attempt to give readers an insight into Sig's personality. We hope we can offer those who knew him through his work a glimpse of how it felt to interact with this remarkable individual.

Comparison of microbial changes in early redeveloping biofilms on natural teeth and dentures.

BACKGROUND: Surfaces and fluids can affect oral bacterial colonization. The aim of this study is to compare redeveloping biofilms on natural teeth and dentures. METHODS: Supragingival plaque samples were taken from 55 dentate individuals and the denture teeth of 62 edentulous individuals before and after professional cleaning. Also, samples from seven "teeth" (samples included dentures) in randomly selected quadrants were collected after 1, 2, 4, and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Counts and proportions of 41 bacterial taxa were determined at each time point, and significant differences were determined using the Mann-Whitney U test. Ecological succession was determined using a modified moving window analysis. RESULTS: Mean total DNA probe counts were similar precleaning but were higher in dentate individuals at all post-cleaning visits (P <0.01). Precleaning edentate biofilms had higher counts and proportions of Streptococcus mitis, Streptococcus oralis, and Streptococcus mutans, whereas dentate individuals had higher proportions of Tannerella forsythia, Selenomonas noxia, and Neisseria mucosa. By day 2, mean counts of all taxa were higher in natural teeth, and most remained higher at day 7 (P <0.01). Succession was more rapid and complex in dentate individuals. Both groups demonstrated increased proportions of S. mitis and S. oralis by day 1. N. mucosa, Veillonella parvula, and Eikenella corrodens increased in both groups, but later in samples from edentate individuals. CONCLUSIONS: "Mature" natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm redevelopment is more rapid and more complex on natural teeth than on denture teeth.

Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization.

OBJECTIVE: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects. MATERIALS AND METHODS: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively. RESULTS: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples. CONCLUSIONS: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals.

Subgingival microbiota in adult Down syndrome periodontitis.

BACKGROUND AND OBJECTIVE: The subgingival microbiota in Down syndrome and non-Down syndrome adults receiving periodic dental care was examined for 40 bacterial species using checkerboard DNA-DNA hybridization and the results were related to clinical periodontal attachment loss. MATERIAL AND METHODS: A total of 44 Down syndrome, 66 non-Down syndrome mentally retarded and 83 mentally normal adults were clinically evaluated. This involved, for each subject, the removal of subgingival specimens from three interproximal sites on different teeth; all subgingival samples per subject were then pooled and assessed for the presence and levels of 40 bacterial species using species-specific whole-genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject attachment loss within Down syndrome and non-Down syndrome subject groups were quantified using Pearson correlation and multiple linear regression analysis. RESULTS: Down syndrome subjects exhibited greater attachment loss than non-Down syndrome subjects (p=0.05). Most microbial species were present in Down syndrome subjects at levels similar to non-Down syndrome subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis in Down syndrome subjects compared with non-Down syndrome study subjects, higher proportions of Treponema socranskii in Down syndrome subjects compared with non-Down syndrome mentally retarded subjects, and higher proportions of Streptococcus constellatus in Down syndrome subjects compared with mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than Down syndrome subjects with no periodontitis (p=0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum ssp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r=0.35-0.42) and higher proportions of Actinomyces naeslundii II and Actinomyces odontolyticus showed negative correlations (r=-0.36 to -0.40), with increasing mean subject attachment loss in Down syndrome adults. CONCLUSION: Individuals with Down syndrome show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in Down syndrome individuals and raise the question as to the reason for increased colonization in Down syndrome.

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

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.

RNA-oligonucleotide quantification technique (ROQT) for the enumeration of uncultivated bacterial species in subgingival biofilms.

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.

Microbiota of deciduous endodontic infections analysed by MDA and Checkerboard DNA-DNA hybridization.

AIMS: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). METHODOLOGY: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples. RESULTS: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) µg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. CONCLUSION: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected.

The microbiota on different oral surfaces in healthy children.

INTRODUCTION: Knowledge of the early oral colonization patterns could provide a better understanding of oral biofilm development and disease initiation that in turn could be the basis for early preventive programmes. METHODS: Microbial samples were collected from five different oral habitats from a total of 93 children (age 3-12 years), attending the Dental School of the University of Athens, who were split into three age groups. A total of 38 microbial species were sought out by the checkerboard DNA-DNA hybridization technique. RESULTS: All of the test species, except Parvimonas micra and Porphyromonas gingivalis, differed significantly among sample locations providing quite distinct microbial profiles for the different oral surfaces. Supragingival and subgingival plaque had similar profiles and exhibited higher proportions of Actinomyces species and Green complex while soft tissue samples were dominated by streptococci of the Yellow complex. The profiles of the tongue dorsum and saliva were also similar. Many of the species were in similar proportions in all three age groups for a given location. Periodontal pathogens showed increases in proportions with increasing age. Specifically, the Red complex species (Tannerella forsythia, P. gingivalis, Treponema denticola) showed a significant increase in proportion with age (P < 0.05) in all sample locations. CONCLUSIONS: The results showed a pattern of colonization in children similar to that previously found in adults. Differences in the profile between age groups suggest a gradual maturation of the oral microbiota, with it being made up of an increasing number of Orange and Red complex species.

Salivary cytokine levels in subjects with chronic periodontitis and in periodontally healthy individuals: a cross-sectional study.

BACKGROUND AND OBJECTIVE: Saliva has been proposed as a noninvasive diagnostic fluid that could be used in the diagnosis of oral and systemic diseases. The levels of salivary biomarkers, such as cytokines, could potentially be used as a surrogate to distinguish periodontally healthy individuals from subjects with periodontitis. Therefore, the goal of the present investigation was to determine if the levels of 10 different cytokines in saliva differed between a group of periodontally healthy individuals and a group of subjects with periodontitis. Correlations between the concentrations of these 10 cytokines and clinical parameters of periodontal disease were also examined. MATERIAL AND METHODS: In this cross-sectional study, 74 subjects with chronic periodontitis and 44 periodontally healthy individuals were periodontally examined and had the levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha measured in whole saliva using a multiplexed bead immunoassay (Luminex). Significance of statistical differences in the levels of salivary cytokines between groups was determined using nonparametric analysis of covariance, adjusting for age and smoking status. The Spearman rank correlation coefficient was used to explore associations between the mean levels of salivary cytokines and mean clinical parameters. RESULTS: There were no statistically significant differences between groups for any of the cytokines. There were weak, statistically significant positive associations between salivary interleukin-8 and pocket depth (r(s) = 0.2, p < 0.05) and bleeding on probing (r(s) = 0.2, p < 0.05), and weak negative correlations between salivary interleukin-10 and attachment level (r(s) = -0.2, p < 0.05) and bleeding on probing (r(s) = -0.3, p < 0.001). CONCLUSION: Mean salivary levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha could not discriminate between periodontal health and disease.

Factors affecting human supragingival biofilm composition. II. Tooth position.

BACKGROUND AND OBJECTIVE: Little is known regarding the factors that affect the microbial composition of supragingival biofilms. This study was designed to test the hypothesis that tooth location affects the microbial composition of supragingival plaque beyond the effect due to plaque mass as reflected by total DNA probe count. MATERIAL AND METHODS: Supragingival plaque samples were taken from the mesiobuccal aspect of each tooth in 187 subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Significance of differences in mean species counts and proportions were determined among tooth surfaces and six tooth type categories: molars, bicuspids, incisors/canines in the mandible and maxilla separately using the Kruskal-Wallis test. Stepwise multiple linear regression was employed to examine the relationship between species proportions and total DNA probe count, tooth location, periodontal and smoking status, age and sex. RESULTS: All species differed significantly among tooth types and among the six tooth categories. Higher plaque levels were seen on molars and lower incisors. Some differences observed between tooth types could be partly explained by the level of plaque. Teeth with high plaque mass exhibited high levels of Capnocytophaga gingivalis, Actinomyces naeslundii genospecies 2, Campylobacter rectus and Campylobacter showae. However, certain species, such as Veillonella parvula and Streptococcus sanguinis, differed significantly at different tooth locations despite similarities in plaque mass. Twenty of the test species exhibited a significant association with tooth location after adjusting for total DNA probe count and subject level factors. CONCLUSION: While plaque mass was associated with differences in proportions of many species in supragingival biofilms, tooth location also was strongly associated with species proportions in both univariate and multivariate analyses.

Factors affecting human supragingival biofilm composition. I. Plaque mass.

BACKGROUND AND OBJECTIVE: Little is known about the factors that affect the microbial composition of supragingival biofilms. This study was designed to examine the relationship between total DNA probe counts of supragingival biofilm samples, clinical parameters and supragingival biofilm composition. MATERIAL AND METHODS: Supragingival plaque samples were taken from 187 systemically healthy adult subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. The relationship between total DNA probe counts and microbial composition was examined by subsetting the data into 10 groups based on 10 percentile increments of the total DNA probe counts. Differences among groups in terms of species counts and proportions were sought, as well as relationships of total plaque DNA probe count and clinical parameters. RESULTS: There was a wide distribution in mean total DNA probe counts among the 187 subjects. With increasing total plaque levels there was a change in the proportions of individual species and microbial complexes. 'Small plaques' were characterized by high proportions of species in the yellow, orange, purple and 'other' complexes; plaques of moderate mass were characterized by high proportions of Actinomyces and purple complex species, while 'large plaques' exhibited increased proportions of green and orange complex species. Measures of gingival inflammation, pocket depth and recession were significantly positively associated with total DNA probe counts. Increased plaque numbers were related to increased pocket depth irrespective of presence or absence of gingival inflammation. CONCLUSION: The proportions of individual species and microbial complexes in supragingival biofilms are influenced by the total numbers of organisms in the biofilm.

Disease progression in periodontally healthy and maintenance subjects.

BACKGROUND: The aim of this study was to determine whether the rate of attachment loss in periodontally healthy subjects in a prevention regimen would differ from the rate of disease progression in periodontitis subjects enrolled in a maintenance program. METHODS: Fifty-five periodontally healthy subjects and 57 periodontitis subjects were clinically and microbiologically monitored at baseline and at 1, 2, and 3 years. Clinical parameters measured at six sites per tooth included bleeding on probing, visible plaque, probing depth, and attachment level. Subgingival plaque samples were taken from the mesio-buccal aspect of every tooth and were analyzed for the levels of 40 bacterial species using checkerboard DNA-DNA hybridization. The significance of differences over time in the clinical parameters was determined using repeated-measures analysis of variance, whereas the significance of differences between groups was determined using the unpaired t test. The Mann-Whitney test was used for microbial analyses, and P values were adjusted for multiple comparisons. RESULTS: Mean clinical parameters improved for both groups over time. By the end of the study, 4% of the sites in maintenance subjects lost > or =2 mm of attachment, whereas in the prophylaxis subjects only 1% of the sites lost > or =2 mm of attachment. Maintenance subjects lost attachment primarily at shallow buccal and lingual sites. The maintenance subjects harbored significantly higher levels of most test species throughout the study. The maintenance program did not reduce the levels of red complex species to those typical of healthy subjects. CONCLUSIONS: Treated periodontitis subjects under maintenance displayed more rapid attachment loss than periodontally healthy subjects in a preventive regimen. The greater propensity to disease progression may be related to an elevated exposure to periodontal pathogens.

Microbial complexes in supragingival plaque.

BACKGROUND/AIMS: To examine microbial communities in supragingival biofilm samples. METHODS: Supragingival plaque samples were taken from 187 subjects at baseline (n = 4745). Fifty-five subjects provided supragingival plaque samples at 1-7 days after professional tooth cleaning (n = 1456); 93 subjects provided 8044 samples between 3 and 24 months post-therapy. All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Microbial associations among species were sought using cluster analysis and community ordination techniques for the three groups separately. RESULTS: Six complexes were formed for the baseline samples. Similar complexes were formed for the samples taken 3-24 months post-therapy. However, distinct changes were observed in microbial communities in samples taken during the 7 days of plaque redevelopment. The complexes related to clinical parameters of periodontal disease. CONCLUSION: There were specific microbial complexes in supragingival plaque that were similar to those found in subgingival plaque samples with a few minor differences. The relation of previously unclustered taxa to the complexes was also described.

Microbiological changes associated with four different periodontal therapies for the treatment of chronic periodontitis.

BACKGROUND/AIMS: To examine subgingival microbiological changes in chronic periodontitis subjects receiving scaling and root planing (SRP) alone or with systemically administered azithromycin, metronidazole or a sub-antimicrobial dose of doxycycline. METHODS: Ninety-two periodontitis subjects were randomly assigned to receive SRP alone or combined with azithromycin, metronidazole or sub-antimicrobial dose doxycycline. Subgingival plaque samples taken at baseline, 2 weeks, and 3, 6, and 12 months were analyzed for 40 bacterial species using checkerboard DNA-DNA hybridization. Percentage of resistant species and percentage of sites harboring species resistant to the test antibiotics were determined at each time-point. RESULTS: All treatments reduced counts of red complex species at 12 months, although no significant differences were detected among treatment groups for most species at all time-points. Both antibiotics significantly reduced counts of red complex species by 2 weeks. Percentage of resistant isolates increased in plaque samples in all adjunctive treatment groups, peaking at the end of administration, but returned to pretreatment levels by 12 months. CONCLUSION: The significant reduction of red and orange complex species at 2 weeks in the subjects receiving SRP plus azithromycin or metronidazole may have contributed to a better clinical response in these treatment groups. Therapy did not appear to create lasting changes in the percentage of resistant isolates or sites harboring resistant species.

Increased interleukin-18 in gingival crevicular fluid from periodontitis patients.

INTRODUCTION: This study aimed to measure the levels of interleukin-18 (IL-18) in inflamed shallow sites and inflamed deep sites in patients with periodontitis and to compare the data with results from inflamed shallow sites in patients with gingivitis. A secondary aim was to examine the composition of the subgingival microbiota in the sampled sites. METHODS: Gingival crevicular fluid was collected from five gingivitis sites and five periodontitis sites from 18 patients with chronic periodontitis, and from five gingivitis sites from 15 patients with gingivitis. Samples from each site category were pooled and IL-18 levels were measured using an enzyme-linked immunosorbent assay. The subgingival microbiota was analyzed by checkerboard DNA-DNA hybridization. RESULTS: All clinical parameters and gingival crevicular fluid volumes were higher in periodontitis sites compared with gingivitis sites from patients with periodontitis and gingivitis. The total amount of IL-18 was higher in periodontitis sites than gingivitis sites in both periodontitis (P = 0.018) and gingivitis (P = 0.002) patients and was higher in gingivitis sites from periodontitis patients than in those from gingivitis patients (P = 0.015). There were higher levels of Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (red complex species) in periodontitis sites compared with gingivitis sites in both the periodontitis and gingivitis patients (P < 0.001). CONCLUSION: Levels of IL-18 were higher in patients with chronic periodontitis compared with patients with gingivitis, even at sites with similar pocket depths. The presence of similar levels of red complex species in gingivitis sites from periodontitis patients and from gingivitis patients suggested that the higher levels of IL-18 were not associated with a different microbial challenge.

Intra-oral microbial profiles of beagle dogs assessed by checkerboard DNA-DNA hybridization using human probes.

Some investigators suggest a similarity between the oral microbiota of dogs and humans. The in vivo assessment of ecologic relationships among bacterial species and between bacterial species and their habitat is difficult to carry out. Consequently, this aspect is often neglected in animal oral microbiological studies. This study aimed to examine the proportions of 40 bacterial species in samples from five intra-oral habitats in beagle dogs using checkerboard DNA-DNA hybridization. Microbial samples were taken from subgingival and supra-gingival plaque, the tongue, tonsils and cheek mucosa in seven beagle dogs. Samples were individually evaluated for their content of 40 bacterial species and the percentage of total DNA probe count was determined for each species, at each habitat. All tested species could be detected in all sampled habitats but each habitat had a distinct community structure. The microbiotas colonizing the hard surfaces in the oral cavity were quite different from the microbiotas colonizing the soft tissues. Bacterial species that are in humans considered to be periodontopathogens are present in high proportions. This study underlines the importance of the habitat and the host on the local microbial profile.

Guiding periodontal pocket recolonization: a proof of concept.

The complexity of the periodontal microbiota resembles that of the gastro-intestinal tract, where infectious diseases are treatable via probiotics. In the oropharyngeal region, probiotic or replacement therapies have shown some benefit in the prevention of dental caries, otitis media, and pharyngitis, but their effectiveness in the treatment of periodontitis is unknown. Therefore, this study addressed the hypothesis that the application of selected beneficial bacteria, as an adjunct to scaling and root planing, would inhibit the periodontopathogen recolonization of periodontal pockets. Analysis of the data showed, in a beagle dog model, that when beneficial bacteria were applied in periodontal pockets adjunctively after root planing, subgingival recolonization of periodontopathogens was delayed and reduced, as was the degree of inflammation, at a clinically significant level. The study confirmed the hypothesis and provides a proof of concept for a guided pocket recolonization (GPR) approach in the treatment of periodontitis.

Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

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 degrees C for 3 min and cooled to 4 degrees C. Phi29 DNA polymerase was added and the mixture was incubated at 30 degrees 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 microg. 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.

A three-year prospective study of adult subjects with gingivitis II: microbiological parameters.

AIM: To investigate whether the clinical benefits obtained with a periodontal prevention programme in subjects with periodontal health or minimal disease were accompanied by beneficial changes in the subgingival microbiota. MATERIAL AND METHODS: One hundred and twenty-four subjects completed the study. Subjects were clinically and microbiologically monitored at baseline, 1, 2 and 3 years. Subgingival plaque samples were taken from the mesiobuccal aspect of every tooth and were analysed for the levels of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples=13,477). The mean counts of each of the 40 test species were calculated for each subject at each time point. Significance of differences over time was sought using the Friedman test. p values were adjusted for multiple comparisons. RESULTS: All clinical parameters, at the microbiologically sampled sites, improved over time. The clinical changes were accompanied by statistically significant decreases in the mean counts of 35 of the 40 test species. Major reductions occurred by year 2 for Actinomyces, Capnocytophaga, Campylobacter, Fusobacterium and Prevotella species. At year 3, there was a modest re-growth of the majority of the species. CONCLUSIONS: The clinical improvements obtained through preventive measures were accompanied by a shift to a more host-compatible subgingival microbiota.