It has been recognized for some time that bacterial species exist in complexes in subgingival plaque. The purpose of the present investigation was to attempt to define such communities using data from large numbers of plaque samples and different clustering and ordination techniques. Subgingival plaque samples were taken from the mesial aspect of each tooth in 185 subjects (mean age 51 +/- 16 years) with (n = 160) or without (n = 25) periodontitis. The presence and levels of 40 subgingival taxa were determined in 13,261 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments were made at 6 sites per tooth at each visit. Similarities between pairs of species were computed using phi coefficients and species clustered using an averaged unweighted linkage sort. Community ordination was performed using principal components analysis and correspondence analysis. 5 major complexes were consistently observed using any of the analytical methods. One complex consisted of the tightly related group: Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola. The 2nd complex consisted of a tightly related core group including members of the Fusobacterium nucleatum/periodonticum subspecies, Prevotella intermedia, Prevotella nigrescens and Peptostreptococcus micros. Species associated with this group included: Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis. The 3rd complex consisted of Streptococcus sanguis, S. oralis, S. mitis, S. gordonii and S. intermedius. The 4th complex was comprised of 3 Capnocytophaga species, Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a. The 5th complex consisted of Veillonella parvula and Actinomyces odontolyticus. A. actinomycetemcomitans serotype b, Selenomonas noxia and Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the 5 major complexes. The 1st complex related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.
Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.
The purpose of the present investigation was to examine the effect of SRP on clinical and microbiological parameters in 57 subjects with adult periodontitis (mean age 47 +/- 11 years). Subjects were monitored clinically and microbiologically prior to and 3, 6 and 9 months after full-mouth SRP under local anaesthesia. Clinical assessments of plaque, redness, suppuration, BOP, pocket depth and attachment level were made at 6 sites per tooth. The means of duplicate attachment level measurements taken at each visit were used to assess change between visits. Clinical data were averaged within each subject and then averaged across subjects for each visit. 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 at each visit. Differences in clinical and microbiological parameters before and after SRP were sought using the Wilcoxon signed ranks test or the Quade test for more than 2 visits. Overall, there was a mean gain in attachment level of 0.11 +/- 0.23 mm (range -0.53 to 0.64 mm) 3 months post-therapy. There was a significant decrease in the % of sites exhibiting gingival redness (68 to 57%) and BOP (58 to 52%) as well as a mean (+/-SEM) pocket depth (3.3 +/- 0.06 to 3.1 +/- 0.05 mm). Sites with pre-therapy pocket depths of < 4 mm showed a non-significant increase in pocket depth and attachment level, 4.6 mm pockets showed a significant decrease in pocket depth and a non-significant gain in attachment post-therapy, while > 6 mm pockets showed a significant decrease in pocket depth and attachment level measurements post-therapy. Significant clinical improvements were seen in subjects who had never smoked or were past smokers but not in current smokers. Mean prevalences and levels of P. gingivalis, T. denticola and B. forsythus were significantly reduced after SRP, while A. viscosus showed a significant increase in mean levels. The mean decrease in prevalence of P. gingivalis was similar at all pocket depth categories, while B. forsythus decreased more at shallow and intermediate pockets and A. viscosus increased most at deep sites. P. gingivalis. B. forsythus and T. denticola were equally prevalent among current, past and never smokers pre-therapy, decreased significantly post-SRP in never and past smokers but increased in current smokers. Clinical improvement post-SRP was accompanied by a modest change in the subgingival microbiota, primarily a reduction in P. gingivalis, B. forsythus and T. denticola, suggesting potential targets for therapy and indicating that radical alterations in the subgingival microbiota may not be necessary or desirable in many patients.
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.
The purpose of the present investigation was to relate clinical characteristics at a site to the frequency of detection, absolute counts and proportions of 14 subgingival species. Subgingival plaque samples were removed by curette from the mesial surface of 2299 teeth in 3 healthy and 87 subjects with periodontal attachment loss. Samples were dispersed, diluted and plated on Trypticase soy agar supplemented with 5% sheep blood. After 7 days of anaerobic incubation, colonies were lifted onto nylon filters, lysed and the DNA fixed to the filters. Digoxygenin-labeled DNA probes were used to identify colonies of each test species. Measurements of pocket depth, attachment level, recession, redness, bleeding on probing and suppuration were made at each sampled site. Total viable counts at sites ranged from 10(3) to greater than 10(8) and were strongly related to pocket depth. Mean total counts at sites less than 3 mm averaged 4.6 x 10(6), while mean counts at sites greater than 7 mm averaged 2.0 x 10(7). Species enumerated and % of sites colonized were as follows; V. parvula 44; S. sanguis II 36; B. intermedius I 33; C. ochracea 31; B. intermedius II 30; S. sanguis I 29; B. gingivalis 27; S. intermedius 25; P. micros 24; W. recta 23; F. nucleatum ss vincentii 18; B. forsythus 15; A. actinomycetemcomitans serotype a 10; A. actinomycetemcomitans serotype b 8. Counts of B. intermedius II were higher at sites which exhibited gingival redness while B. intermedius I was higher at sites which bled on probing. A. actinomycetemcomitans serotype b was more frequent and at higher mean % at sites without recession. The opposite was true for S. sanguis II. B. gingivalis was somewhat more prevalent and at higher levels at suppurating sites. B. gingivalis, B. intermedius I and II and B. forsythus were found more frequently and at higher levels at sites with deeper pockets, while V. parvula was less prevalent at sites with pocket depths less than 4 mm. B. gingivalis, B. intermedius I and A. actinomycetemcomitans serotype b increased with increasing pocket depth in both localized and widespread disease subjects, but mean counts were higher in the localized disease subjects at any pocket depth. Only W. recta was found at higher levels at deep sites in widespread disease subjects when compared with similar sites in localized disease subjects. No suspected pathogens were detected in 38% of shallow sites, 31% of intermediate sites and 22% of deep sites, 2/3 of deep pockets, but less than 1/2 of shallow pockets harbored at least 2 of the suspected pathogens.
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