Characterizing Microbiota from Sjögren's Syndrome Patients.

OBJECTIVE: To compare the oral microbiota of Sjögren's syndrome (SS) with that of healthy subjects (HS). METHODS: Supragingival and subgingival biofilm samples were collected from the mesial-buccal tooth surfaces of SS patients (n = 57) and age- and sex-matched HS (n = 53). Unstimulated saliva and 8 oral tissue samples were taken using a buccal brush. Caries and periodontal measures were recorded. All supragingival samples and a subgroup of 24 SS and 28 HS subgingival samples, as well as 32 SS and 11 HS saliva and oral tissue samples, were analyzed for their content of 41 bacterial species using checkerboard DNA-DNA hybridization. Mean levels (×105 ± SEM) and percentage of DNA probe counts of each species were determined for each sample site and averaged within subjects in the 2 clinical groups. Kruskal-Wallis tests, adjusting for multiple comparisons and cluster analysis, were used for soft tissue and microbial analysis, and the Mann-Whitney test was used to compare caries and periodontal measures. RESULTS: Mean (×105 ± SEM) total DNA probe counts in supragingival samples were significantly lower (P < 0.001) in the SS (13.3 ± .7) compared to the HS (44.1 ± 6.8) group. In supragingival samples, Veillonella parvula, Fusobacterium nucleatum ss vincenti, and Propionibacterium acnes were markedly elevated in the SS compared to the HS group in both mean (×105 ± SEM) and mean (± SEM) percentage DNA probe counts (P < 0.001). In subgingival samples of SS, V. parvula was significantly different compared to HS (P < 0.05). SS was characterized by high levels of purple and low levels of orange and red complexes. Cluster analysis of oral tissues and saliva demonstrated that the mean microbial profiles for SS patients and the HS group clustered separately. Active root caries (P < 0.003) and attachment loss were significantly higher (P < 0.029) in the SS group compared to the HS group. CONCLUSION: These findings indicate that saliva is a major controlling factor of intraoral biofilm. V. parvula may be a unique microbial biomarker for Sjögren's syndrome. KNOWLEDGE TRANSFER STATEMENT: The microbiome characterized for Sjögren's syndrome in salivary hypofunction is shown to be under stress and reduced. Veillonella parvula can be a possible identification of a biomarker for Sjögren's syndrome.

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.

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.

Initial subgingival colonization of 'pristine' pockets.

The treatment of periodontitis/peri-implantitis involves the reduction/eradication of periopathogens. After therapy, beneficial and pathogenic species recolonize the subgingival area. The dynamics of recolonization and especially the role of the supragingival environment in this process are still not well-understood. This prospective, split-mouth study followed the early colonization of 'pristine' pockets created during implant surgery (16 partially edentulous patients), to record the time needed before a complex subgingival flora could be established with the supragingival area as the single source. Four subgingival plaque samples were taken from shallow and medium pockets around implants (test), and neighboring teeth (undisturbed microbiota as reference) 1, 2, and 4 wks after abutment connection. Checkerboard DNA-DNA hybridization and culture data revealed a complex microbiota (including several pathogenic species) in the pristine pockets within a wk, with a minimal increase in counts up to 4 wks. Analysis of these data demonstrated that, even with the supragingival environment as the single source for colonizing bacteria, a complex subgingival microbiota can develop within 1 wk.

Use of checkerboard DNA-DNA hybridization to study complex microbial ecosystems.

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.

Clinical and microbiological changes associated with the use of combined antimicrobial therapies to treat "refractory" periodontitis.

BACKGROUND: The present investigation examined clinical and microbial changes after a combined aggressive antimicrobial therapy in subjects identified as "refractory" to conventional periodontal therapy. METHOD: Fourteen subjects were identified as "refractory" based on full-mouth mean attachment loss and/or >3 sites with attachment loss > or =3 mm following scaling and root planing (SRP), periodontal surgery and systemic antibiotics. After baseline monitoring, subjects received SRP, locally delivered tetracycline at pockets > or =4 mm, systemically administered amoxicillin (500 mg, t.i.d. for 14 days)+metronidazole (250 mg, t.i.d. for 14 days) and professional removal of supragingival plaque weekly for 3 months. Subjects were monitored clinically every 3 months post-therapy for 2 years. Subgingival plaque samples were taken at the same time points from the mesial aspect of each tooth and the levels of 40 subgingival taxa were determined using checkerboard DNA-DNA hybridization. Mean levels of each species were averaged within a subject at each visit. Significance of changes in clinical and microbiological parameters over time were evaluated using the Friedman or Wilcoxon signed ranks test. RESULTS: On average, subjects showed significant improvements in all clinical parameters after therapy. Mean (+/-SEM) full-mouth pocket depth reduction was 0.83+/-0.13 mm and mean attachment level "gain" was 0.44+/-0.12 at 24 months. Clinical improvement was accompanied by major reductions in multiple subgingival species during the first 3 months of active therapy that were maintained for most species to the last monitoring visit. Reductions occurred for three Actinomyces species, "orange complex" species including Campylobacter showae, Eubacterium nodatum, three Fusobacterium nucleatum subspecies, Peptostreptococcus micros, Prevotella intermedia as well as the "Streptococcus milleri" group, Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedus. Subjects differed in their response to therapy; six modest response subjects exhibited less attachment level gain and were characterized by reductions in the microbiota from baseline to 3 months, but re-growth of many species thereafter. CONCLUSIONS: The combined antibacterial therapy was successful in controlling disease progression in 14 "refractory" periodontitis subjects for 2 years.

Relationship between periodontal pocket sulfide levels and subgingival species.

BACKGROUND: Many species implicated in the pathogenesis of periodontal disease produce volatile sulfur compounds (VSC). This investigation examined the relationship between levels of sulfide and subgingival bacterial species in the same periodontal pockets. MATERIAL AND METHODS: Twenty chronic periodontitis subjects were measured clinically at six sites per tooth for plaque, gingivitis, bleeding on probing, suppuration, pocket depth and attachment level. Subgingival plaque samples, taken from the mesial aspect of each tooth, were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Sulfide levels were measured at the same sites using a Diamond Probe/Perio 2000 system. Clinical and microbiological data were averaged for sulfide-positive and -negative sites separately in each subject and then averaged across subjects. Significance differences in clinical and microbial parameters between sulfide-positive and -negative sites were sought using the Wilcoxon signed ranks test. RESULTS: Mean total DNA probe counts (x10(5), +/-SEM) at sulfide-negative and -positive sites were 44.0 +/- 9.9 and 65.0 +/- 13.3, respectively (p < 0.01). Seventeen species were found at significantly higher levels in sulfide-positive than -negative sites. These included abundant producers of VSC such as members of the genera Fusobacterium, Campylobacter, Prevotella, Treponema and Eubacterium, and Bacteriodes forsythus, Selenomonas noxia and Propionibacterium acnes. Prevotella intermedia, Bacteriodes forsythus, Prevotella nigrescens, Fusobacterium nucleatum ss vincentii and Treponema denticola exhibited the greatest difference in mean counts between sulfide-negative and -positive sites. Orange and red complex species were at higher counts at shallow (< 4 mm) sulfide-positive than shallow sulfide-negative sites. Although not statistically significant, mean clinical parameters were somewhat higher at sulfide-positive than sulfide-negative sites. CONCLUSIONS: Intra-pocket sulfide levels reflect the levels of sulfide-producing species and may provide useful diagnostic information.