Aging and Oral Health: Biological and Sociobehavioral Perspectives.

As the life expectancy and growth of the aging population increase globally, efforts to promote healthy longevity become more important. Holistic policy guidelines and actions have been designed to advocate and fortify healthy aging at multiple levels. Oral health, a fundamental contributor of overall health and well-being, forms a core part of the noncommunicable disease agenda within the sustainable development goals set by the World Health Organization. Aging significantly heightens the risk of myriad oral disorders and other noncommunicable diseases. As of 2019, oral disorders accounted for 8.9 million disability-adjusted life-years in individuals older than 60 y. In addition to the development of multidisciplinary aging-friendly policies to promote healthy aging, basic biology and translational research has been encouraged that focuses on deciphering the underlying mechanisms involved in age-related physical and cognitive decline or dysregulation of oral tissues. Given the relevance of oral health aging as a critical component of the One Health Initiative, this special issue encompasses a collection of articles dedicated to recent advances in the behavioral and social implications of age-related oral diseases and tooth loss on several aspects of the quality of life of adults as they age. Furthermore, it includes articles detailing molecular mechanisms associated with cellular aging and their implications for oral tissue health, periodontal disease severity, and the regenerative potential of stem cells.

The Role of Bacterial Biofilms in Dental Caries and Periodontal and Peri-implant Diseases: A Historical Perspective.

Over the last hundred years, groundbreaking research in oral microbiology has provided a broad and deep understanding about the oral microbiome, its interactions with our body, and how the community can affect our health, be protective, or lead to the development of dental diseases. During this exciting journey, hypotheses were proposed, and concepts were established, discarded, and later revisited from updated perspectives. Dental plaque, previously considered a polymicrobial community of unspecific pathogenicity, is recognized as microbial biofilms with healthy, cariogenic, or periodontopathogenic profiles, resulting from specific ecologic determinants and host factors. The "one pathogen, one disease" paradigm of oral infections has been replaced by a holistic concept of a microbial community as the entity of pathogenicity. Cutting-edge technology can now explore large microbial communities related to different clinical conditions, which has led to finding several novel disease-associated species and potential pathobionts and pathobiomes. This vast amount of data generated over time has widened our view of the etiology of caries and periodontal and peri-implant diseases and has promoted updated strategies to treat and prevent the oral diseases.

Clinical and microbiological parameters of naturally occurring periodontitis in the non-human primate Macaca mulatta.

Background: Non-human primates appear to represent the most faithful model of human disease, but to date the oral microbiome in macaques has not been fully characterized using next-generation sequencing. Objective: In the present study, we characterized the clinical and microbiological features of naturally occurring periodontitis in non-human primates (Macaca mulatta). Design: Clinical parameters of periodontitis including probing pocket depth (PD) and bleeding on probing (BOP) were measured in 40 adult macaques (7-22 yrs), at six sites per tooth. Subgingival plaque was collected from diseased and healthy sites, and subjected to 16S rDNA sequencing and identification at the species or higher taxon level. Results: All macaques had mild periodontitis at minimum, with numerous sites of PD ≥ 4 mm and BOP. A subset (14/40) had moderate-severe disease, with >2 sites with PD ≥ 5mm, deeper mean PD, and more BOP. Animals with mild vs moderate-severe disease were identical in age, suggesting genetic heterogeneity. 16S rDNA sequencing revealed that all macaques had species that were identical to those in humans or closely related to human counterparts, including Porphyromonas gingivalis which was present in all animals. Diseased and healthy sites harboured distinct microbiomes; however there were no significant differences in the microbiomes in moderate-severe vs. mild periodontitis. Conclusions: Naturally occurring periodontitis in older macaques closely resembles human adult periodontitis, thus validating a useful model to evaluate novel anti-microbial therapies.

Subgingival bacterial community profiles in HIV-infected Brazilian adults with chronic periodontitis.

BACKGROUND AND OBJECTIVE: To compare the subgingival microbial diversity between non-HIV-infected and HIV-infected individuals with chronic periodontitis using denaturing gradient gel electrophoresis (DGGE). MATERIAL AND METHODS: Thirty-two patients were selected: 11 were HIV-infected and 21 were non-HIV-infected, and all had chronic periodontitis. Periodontal measurements included probing depth, clinical attachment level, visible supragingival biofilm and bleeding on probing. Subgingival biofilm samples were collected from periodontal sites (50% with probing depth ≤ 4 mm and 50% with probing depth ≥ 5 mm) and whole-genomic-amplified DNA was obtained. The DNA samples were subjected to amplification of a 16S rRNA gene fragment using universal bacterial primers, followed by DGGE analysis of the amplified gene sequences. RESULTS: The non-HIV-infected group presented higher mean full-mouth visible supragingival biofilm (p = 0.004), bleeding on probing (p = 0.006), probing depth (p < 0.001) and clinical attachment level (p = 0.001) in comparison with the HIV-infected group. DGGE analysis revealed 81 distinct bands from all 33 individuals. Banding profiles revealed a higher diversity of the bacterial communities in the subgingival biofilm of HIV-infected patients with chronic periodontitis. Moreover, cluster and principal component analyses demonstrated that the bacterial community profiles differed between these two conditions. High interindividual and intra-individual variability in banding profiles were observed for both groups. CONCLUSION: HIV-infected patients with chronic periodontitis present greater subgingival microbial diversity. In addition, the bacterial communities associated with HIV-infected and non-HIV-infected individuals are different in structure.

Ex vivo antimicrobial efficacy of the EndoVac system plus photodynamic therapy associated with calcium hydroxide against intracanal Enterococcus faecalis.

AIM: To evaluate the ex vivo efficacy of the EndoVac system and photodynamic treatment (PDT) as adjuncts to chemomechanical debridement associated with calcium hydroxide (CaOH2 ) in reducing the levels of intracanal Enterococcus faecalis. METHODOLOGY: One hundred and twenty-five sterile premolar teeth were conventionally accessed, prepared and then contaminated with E. faecalis (ATCC 29212) for 30 days. Teeth were randomly divided into 4 groups: Control (chemomechanical debridement with conventional irrigation); Endovac (chemomechanical debridement with EndoVac system); PDT (chemomechanical debridement with conventional irrigation and PDT) and Endovac+PDT (chemomechanical debridement with EndoVac and PDT). The irrigants used in all groups were 5.25% sodium hypochlorite and 17% EDTA. After treatment, an intracanal dressing (CaOH2 ) was applied in all canals for 7 days. Samples were obtained before (T1) and after the therapeutic procedures (T2) and, after intracanal medication (T3), plated onto BHI media and incubated (37 °C, 48 h) to determine the colony-forming units (CFU mL(-1) ). RESULTS: The overall mean cell counts (CFU mL(-1) ) of E. faecalis were high at the initial contamination (T1). A significant reduction (P < 0.05) of E. faecalis mean counts was observed in all groups from baseline (T1) to both post-therapy samplings (T2 and T3); no differences amongst the groups were detected. No significant change in bacterial counts from T2 to T3 was detected. CONCLUSION: The adjunctive use of the EndoVac system and the photodynamic treatment, in combination or not, was as effective as the conventional chemomechanical debridement associated with CaOH2 on reducing the counts of intracanal E. faecalis.

Detection of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival biofilm of HIV-infected subjects undergoing HAART with chronic periodontitis.

This study compared the frequency of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival microbiota of HIV-seropositive and HIV-seronegative subjects with periodontitis or clinically healthy periodontal tissues. Fifty-four subjects were distributed into two HIV-seropositive groups (chronic periodontitis [HCP = 13] and periodontal health [HH = 10]) and two HIV-seronegative groups (chronic periodontitis [CP = 17] and periodontal health [H = 14]). The detection of bacterial species was carried out by polymerase chain reaction (PCR). CP patients showed significantly more periodontal destruction, inflammation, and supragingival plaque than HCP patients (P < 0.05). All species were detected at a higher prevalence in CP and HCP than H individuals (P < 0.01). In the HIV groups, H. pylori was significantly more prevalent in periodontitis compared to healthy patients (P < 0.01). A higher frequency of E. faecalis and P. aeruginosa was observed in the subgingival biofilm of HH than H subjects (P < 0.01). Moreover, E. faecalis was detected significantly more often in HIV-seropositive compared to HIV-seronegative patients, regardless of periodontal status (P < 0.01). These data indicate that H. pylori is frequently detected in the subgingival microbiota of periodontitis subjects. In contrast, HIV-seropositive patients with either periodontitis or periodontal health present a high prevalence of E. faecalis.

Relationship of neutrophil phagocytosis and oxidative burst with the subgingival microbiota of generalized aggressive periodontitis.

INTRODUCTION: Polymorphonuclear neutrophil (PMN) dysfunctions have been associated with severe forms of periodontitis. This study evaluated the correlation between PMN phagocytosis and oxidative burst with the subgingival microbiota of patients with generalized aggressive periodontitis (GAgP). METHODS: Heparinized peripheral blood samples were obtained from 18 GAgP patients and 11 periodontally healthy (PH) subjects, and PMNs were isolated on a Ficoll-Hypaque gradient. For phagocytosis analysis, PMNs were incubated with fluorescein-labeled Staphylococcus aureus. The oxidative burst was evaluated by incubation of PMNs with dihydroethidium and activation by S. aureus. The assays were examined using flow cytometry. Subgingival biofilm samples were obtained from periodontal sites with and without periodontitis and 24 species were detected by checkerboard. RESULTS: A significantly lower phagocytosis rate was observed for patients with GAgP compared with PH subjects over time (P < 0.05). No differences between groups were found for superoxide production. GAgP patients presented significantly higher prevalence and levels of Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans serotype b than controls (P < 0.05). Significant negative correlations between T. forsythia and P. gingivalis and PMN functions were observed. CONCLUSIONS: GAgP subjects presented diminished phagocytic activity of peripheral PMNs and high prevalence and levels of classical periodontal pathogens.

Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization.

BACKGROUND AND OBJECTIVE: Interactions between oral bacteria and gingival epithelial cells play an important role in the pathogenesis of periodontal diseases. This study used in situ hybridization with 16 rRNA probes and confocal microscopy to detect the periodontal pathogens Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythia, and Treponema denticola within epithelial cells from periodontal pockets, gingival crevice, and buccal mucosa collected from subjects with chronic periodontitis (n = 14) and good periodontal health (n = 8). MATERIAL AND METHODS: Each green fluorescent species-specific and universal probe was hybridized with all 58 epithelial samples from the 22 patients. The samples were observed by confocal microscopy to confirm the intracellular localization of oral species of bacteria. The mean frequency of detection and number of intracellular bacteria per epithelial cell were computed for each sample. RESULTS: The frequency of cells with internalized bacteria was higher in samples from the gingival crevice than in samples from the oral mucosa. Epithelial cells from all subjects harbored intracellular bacteria; however, patients with periodontitis presented significantly higher counts of bacteria per cell than periodontally healthy individuals (p < 0.05). Periodontal pathogens showed a trend to be detected in higher numbers in epithelial cells from periodontitis patients. In particular, T. forsythia and T. denticola were significantly more prevalent in periodontal pocket cells than healthy sulci and buccal cell samples in the periodontitis group (p < 0.05). CONCLUSION: Those findings indicate that crevicular and buccal cells present internalized bacteria, regardless of periodontal status. However, higher bacterial loads are detected in cells from subjects with periodontitis.

Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections.

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).

Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization.

OBJECTIVE: The purpose of the present investigation was to examine the microbiota of acute periradicular abscesses of endodontic origin by using a molecular genetic method. STUDY DESIGN: Pus was collected by aspiration from 27 cases diagnosed as acute abscesses of endodontic origin, and DNA was extracted to evaluate the occurrence of 49 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The presence of bacterial DNA in clinical samples was confirmed by polymerase chain reaction with ubiquitous bacterial 16S rRNA gene primers. RESULTS: The results of the checkerboard DNA-DNA hybridization analysis revealed that 37 of the 49 DNA probes tested were reactive with one or more samples. The number of bacterial species in the pus samples ranged from 1 to 33 (mean, 5.9). Eighteen of the 27 pus samples were positive for at least one DNA probe. The most prevalent species found were: Bacteroides forsythus (29.6% of the cases); Porphyromonas gingivalis (29.6%); Streptococcus constellatus (25.9%), Prevotella intermedia (22.2%), Prevotella nigrescens (22.2%), Fusobacterium periodonticum (18.5%), Fusobacterium nucleatum subspecies nucleatum (18.5%), and Eikenella corrodens (18.5%). CONCLUSIONS: The microbiologic data of the present investigation indicated that molecular genetic methods could provide additional knowledge regarding the microbiota of acute periradicular abscesses by detecting bacterial species that are difficult--or even impossible--to grow.

Project, design, and use of a pilot plant for nanocapsule production.

The aim of this study was to find the scale-up parameters necessary for the preparation of nanocapsules (NCs) for pharmaceutical purposes. Starting from the laboratory scale (0.06 L), we designed and assembled a pilot plant (2 L) to produce NCs with the so-called emulsification-diffusion technique. We wanted to check if classical tools adequate for the pharmaceutical industry and for industrial scale-up purposes according to well-known chemical engineering technique could be used to perform the NC preparation. Experiments were carried out by varying some operative parameters, such as the impeller speed, the agitation duration for the emulsion preparation, and the reagent concentrations. As expected, good accordance between the NC produced at the laboratory scale and at the pilot plant scale was obtained. We conclude that the pilot plant can be used to perform a scale-up study of the industrial production of NC.

Checkerboard DNA-DNA hybridization analysis of endodontic infections.

OBJECTIVE: The purpose of this investigation was to examine the microbiota of infected root canals by using a molecular genetic method. STUDY DESIGN: The presence and levels of 42 bacterial species were determined in 28 root canal samples by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. To confirm the presence of bacterial DNA in clinical samples, a polymerase chain reaction with an ubiquitous bacterial primer was undertaken. RESULTS: The results of the checkerboard DNA-DNA hybridization analysis showed that 22 of the 42 DNA probes tested were reactive with 1 or more samples. The number of bacterial species in the root canal samples ranged from 1 to 17 (mean, 4.7). Seventeen of the 28 root canal samples were positive for at least 1 DNA probe. The most prevalent species found were as follows: Bacteroides forsythus (39. 3% of the cases); Haemophilus aphrophilus (25%); Corynebacterium matruchotii (21.4%); Porphyromonas gingivalis (17.9%); and Treponema denticola (17.9%). CONCLUSIONS: The microbiologic data of the present investigation indicated that molecular genetic methods can provide significant additional knowledge regarding the endodontic microbiota by detecting bacterial species that are difficult or impossible to culture. In addition, our findings support the current concept that endodontic infections are mixed infections of polymicrobial etiology.

Discrimination of refractory periodontitis subjects using clinical and laboratory parameters alone and in combination.

The purpose of the present investigation was to use baseline clinical and laboratory parameters to distinguish subjects refractory to conventional periodontal therapy. Baseline clinical, microbial and host parameters were compared in 61 successfully-treated and 27 refractory subjects. Refractory subjects showed mean full-mouth attachment level (AL) loss and/or >3 sites with new AL loss >2.5 mm within 1 year after both scaling and root planing and surgery with systemic tetracycline. Successfully-treated subjects showed mean AL gain and no sites with new AL loss >2.5 mm after either regimen. Gingival redness, bleeding on probing, suppuration, supragingival plaque accumulation, pocket depth and AL were measured at 6 sites per tooth in each subject. The levels of 40 subgingival taxa were determined in subgingival plaque samples from up to 28 sites in each subject using checkerboard DNA-DNA hybridization. Serum antibody (Ab) to 85 subgingival species was determined using checkerboard immunoblotting. Levels of serum IgG2 and Gm23 allotype were measured using radial immunodiffusion; FcgammaRIIa and FcgammaRIIIb receptor haplotypes were determined using PCR and allele specific oligonucleotide probes. Odds ratios of a subject being refractory were determined by comparing measured parameters in the 2 subject groups using univariate and multivariate techniques. 17 of 151 clinical, microbial and immunological variables were significant using chi2 analysis after adjusting for multiple comparisons. For example, the odds ratios of a subject being refractory were 12.2, 5.4 and 6.9 if the subject had Ab >50 microg/ml to >9 species; S. constellatus counts >2.4% of the total DNA probe count or >2.1% of sites with AL >6 mm. The 17 significant predictor variables were used in logistic regression and discriminant analyses. Similar variables were selected using both analyses including the number of serum Ab to subgingival species >50 microg/ml, % S. constellatus in plaque samples and % sites with attachment loss >6 mm. In the logistic regression analysis model, the odds ratios associated with >9 species exhibiting >Ab 50 microg/ml, >2.1% of sites with AL >6 mm and >2.4% S. constellatus in plaque were 8.7, 6.8 and 2.4, respectively, after adjusting for other variables in the model. Discriminant analysis using these variables provided sensitivity, specificity, positive and negative predictive values of 0.66, 0.92, 0.80 and 0.85 respectively. Refractory periodontitis subjects could be distinguished using a subset of clinical, microbiological and immunological parameters.

Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role.

Some structural features of bovine tryptase were discussed based on spectroscopic analysis. The far UV-CD spectrum of the enzymatically active bovine tryptase is consistent with a structure containing very little, if any alpha-helix, as found for other serine proteases. The analysis of near UV-CD and UV absorption spectra reveals the presence of a high number of Trp residues arranged probably in strong structural motifs. At variance with other tryptases, the bovine enzyme shows an electrophoretic behaviour in native and denaturating conditions compatible with an association state larger than a tetramer (probably a dodecamer). From a biochemical point of view, the bovine tryptase shares with the human counterpart, the preference for cleaving substrates bearing dibasic cleavage sites. Thus, it is hypothesized that tryptase may be involved in some proprotein processing mechanism(s).

Serum antibodies reacting with subgingival species in refractory periodontitis subjects.

The purpose of this investigation was to compare the levels of serum IgG antibody to 85 subgingival species in 32 refractory periodontitis, 56 successfully treated, and 33 periodontally healthy subjects. Refractory subjects showed mean full mouth attachment loss and/or >3 sites showing attachment loss >2.5 mm within 1 year after 2 treatment modalities, scaling and root planing and surgery plus systemically administered tetracycline. Successfully-treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm, 1 year post-therapy. Periodontally healthy subjects exhibited no pocket or attachment level >3 mm, and no evidence of progressing attachment loss during 1 year of monitoring. Baseline serum was obtained from each subject and tested against 85 subgingival species, including reference strains and strains isolated from refractory subjects, using checkerboard immunoblotting. Significance of differences in levels of serum antibody among groups were sought using the Kruskal-Wallis test. Refractory subjects constituted a heterogeneous group based on their serum antibody response to subgingival species. Some individuals had antibody reactions to many subgingival species, while other subjects showed fewer or low numbers of responses. On average, refractory subjects exhibited higher numbers and levels of serum antibody reactions to a wide range of subgingival species than successfully treated or periodontally healthy subjects. Differences in serum antibody among clinical groups were more striking at higher threshold levels of antibody (>50 microg/ml and > 100 microg/ml). The data showed that a subject was 10.1 x more likely to be refractory if the subject exhibited antibody reactions with >9 subgingival species at >50 microg/ml (p<0.001, after adjusting for multiple comparisons). Serum antibody to a subset of the test species differed among the clinical groups. Porphyromonas gingivalis, Bacteroidesforsythus, and some strains isolated from refractory subjects (a novel Neisseria sp., Enterococcus faecalis, Prevotella loescheii and Prevotella oulora) elicited high serum antibody in the successfully treated and refractory subjects. High levels of serum antibody to a Microbacterium lacticum-like organism, Streptococcus oralis, Streptococcus constellatus, Actinobacillus actinonmycetemcomitans serotype c and Haemophilus aphrophilus significantly increased the likelihood of a subject being refractory to conventional periodontal therapy.

Serum IgG2 level, Gm(23) allotype and FcgammaRIIa and FcgammaRIIIb receptors in refractory periodontal disease.

The purpose of this investigation was to compare the levels of serum IgG2, the frequency of detection of Gm(23)-negative allotype and frequency of detection of FcgammaRIIa and FcgammaRIIIb receptor haplotypes in 32 refractory, 54 successfully treated and 27 periodontally healthy individuals. Refractory subjects showed mean full mouth attachment loss and/or >3 sites with attachment loss >2.5 mm within 1 year after both scaling and root planing, and surgery plus systemically administered tetracycline. Successfully treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm 1 year post-therapy. Periodontally healthy subjects exhibited no pocket depth or attachment level >3 mm, and no evidence of progressing disease during 1 year of monitoring. Blood was obtained from each subject at baseline. Serum IgG2 and Gm(23) allotype were determined using radial immunodiffusion. DNA was extracted from whole blood and the FcgammaR genotypes determined using PCR and allele specific oligonucleotide probes. Significance of differences among clinical groups were sought using the Kruskal-Wallis or chi-square tests. Associations between 2 or more variables were tested using regression analysis. Refractory subjects exhibited higher mean attachment loss and pocket depth than successfully treated or periodontally healthy subjects. Smoking status did not differ significantly among groups. No significant differences in serum IgG2 levels and frequency of detection of Gm(23)-negative allotype were observed among the clinical groups. Serum IgG2 level was positively associated with the number of serum antibody responses to subgingival species (r=0.51, p<0.001). Subjects with the Gm(23)-negative allotype exhibited lower mean levels of serum IgG2 (3.06+/-0.3 versus 3.9+/-0.2, p<0.01) and mean number of serum antibodies to subgingival species (17.7+/-1.7 versus 23.3+/-1.4, p<0.05) than allotype positive individuals. No significant differences in FcgammaR haplotype distribution were observed among the 3 clinical groups. Associations of serum IgG2 level, Gm(23) allotype, FcgammaRIIa and FcgammaRIIIb receptor haplotypes and smoking status were weakly related or not related to clinical status. This lack of relationship may have been due to a reality of no relationship, or the inadvertent pooling of subjects where these factors were of primary importance with subjects in whom these factors played a less important role.

Clinical and microbiological features of refractory periodontitis subjects.

The purpose of this investigation was to compare the clinical parameters and the site prevalence and levels of 40 subgingival species in successfully treated and refractory periodontitis subjects. 94 subjects received scaling and root planing and if needed, periodontal surgery and systemically administered tetracycline. 28 refractory subjects showed mean full mouth attachment loss and/or > 3 sites showing attachment loss > 2.5 mm within 1 year post-therapy. 66 successfully treated subjects showed mean attachment level gain and no sites with attachment loss > 2.5 mm. Baseline subgingival plaque samples were taken from the mesial aspect of each tooth and the presence and levels of 40 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The mean levels and % of sites colonized by each species (prevalence) was computed for each subject and differences between groups sought using the Mann-Whitney test. Most of the 40 species tested, including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Bacteroides forsythus, were equally or less prevalent in the refractory group. Prevotella nigrescens was significantly more prevalent in successfully treated subjects, while refractory subjects harbored a larger proportion of Streptococcus species, particularly Streptococcus constellatus. The odds of a subject being refractory was 8.6 (p < 0.001) if S. constellatus constituted > or = 3.5% of the total DNA probe count. Since few microbiological differences existed between treatment outcome groups using DNA probes to known species, the predominant cultivable microbiota of 33 subgingival samples from 14 refractory subjects was examined. 85% of the 1649 isolates were identified using probes to 69 recognized subgingival species. The remaining unidentified strains were classified by analyzing 16S rRNA gene sequences. Many sequenced isolates were of taxa not considered a common part of the oral microbiota such as Acinetobacter baumanni, Gemella haemolysans, Enterococcus faecalis, Staphylococcus warneri, Pseudomonas aeruginosa and novel species in the genera Bartonella, Ralstonia, Neisseria, Eubacterium, Rothia, Gordona, Gemella, Corynebacterium, Leptotrichia, and Actinomyces. Refractory subjects constituted a heterogeneous group based on their subgingival microbiota. As a group, they did not harbor more of the "classic" periodontopathogens, although elevated proportions of S. constellatus were found.