Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT).

OBJECTIVE: The aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome. DESIGN: Total nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis. RESULTS: The automated extraction method at 63°C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363. CONCLUSIONS: In general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites.

Bacterial resistance to minocycline after adjunctive minocycline microspheres during periodontal maintenance: A randomized clinical trial.

BACKGROUND: Despite widespread use, the impact of minocycline hydrochloride microspheres on the shifts of oral bacterial species resistant to minocycline remains unknown. This study aimed at examining the percentage and taxonomy of minocycline-resistant isolates in saliva and subgingival plaque samples before and after minocycline microspheres application in periodontitis patients during maintenance. METHODS: Patients received supra- and sub-gingival debridement with (test) or without (control) minocycline microspheres application to sites with probing depth >4 mm and were clinically monitored at baseline, 1, 3, and 6 months. Samples were collected at baseline, 1 and 6 months and analyzed via cultivation with or without 4 µg/mL minocycline. Percentage of resistant strains was determined by colony counting and taxonomy by checkerboard DNA-DNA hybridization. Significant clinical changes were sought with the Mann-Whitney test and differences in percentage of resistant isolates with the Friedman and Mann-Whitney tests. RESULTS: Groups showed similar clinical improvements. Mean percentage of resistant isolates rose at 1 month and decreased at 6 months in saliva and plaque samples in test group (P <0.05) but remained unchanged in control group. Percentage of resistant isolates of Gemella morbillorum and Eubacterium saburreum increased significantly at 6 months in both groups. Antibiotic resistance by Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Porphyromonas gingivalis was either absent or infrequent. CONCLUSION: Minocycline microspheres result in transient selection of minocycline resistant species in saliva and subgingival plaque samples.

The Microbiome of Peri-implantitis: Is It Unique?

A better characterization of the peri-implant microbiome can improve the understanding of the etiology of peri-implant diseases. Ultimately, more detailed information about the peri-implantitis microbiome will lead to better strategies for prevention, supportive therapy, and risk assessment, as well as early diagnosis of peri-implantitis and timely intervention, all of which are critical for the long-term retention of implants.

Exploring the microbiome of healthy and diseased peri-implant sites using Illumina sequencing.

AIM: To compare the microbiome of healthy (H) and diseased (P) peri-implant sites and determine the core peri-implant microbiome. MATERIALS AND METHODS: Submucosal biofilms from 32 H and 35 P sites were analysed using 16S rRNA sequencing (MiSeq, Illumina), QIIME and HOMINGS. Differences between groups were determined using principal coordinate analysis (PCoA), t tests and Wilcoxon rank sum test and FDR-adjusted. The peri-implant core microbiome was determined. RESULTS: PCoA showed partitioning between H and P at all taxonomic levels. Bacteroidetes, Spirochetes and Synergistetes were higher in P, while Actinobacteria prevailed in H (p < .05). Porphyromonas and Treponema were more abundant in P while Rothia and Neisseria were higher in H (p < .05). The core peri-implant microbiome contained Fusobacterium, Parvimonas and Campylobacter sp. T. denticola, and P. gingivalis levels were higher in P, as well as F. alocis, F. fastidiosum and T. maltophilum (p < .05). CONCLUSION: The peri-implantitis microbiome is commensal-depleted and pathogen-enriched, harbouring traditional and new pathogens. The core peri-implant microbiome harbours taxa from genera often associated with periodontal inflammation.

Relationships among interleukin-6, tumor necrosis factor-α, adipokines, vitamin D, and chronic periodontitis.

BACKGROUND: The aim of this study is to explore relationships among serum adipokines, vitamin D, and clinical and microbial parameters of chronic periodontitis before and after treatment. METHODS: Weight, height, and smoking status were recorded for 56 patients with chronic periodontitis. Plaque, gingivitis, bleeding on probing, suppuration, probing depth, and clinical attachment level were measured at all teeth present. Subgingival biofilm samples from each tooth were analyzed for levels of 40 bacterial species using checkerboard DNA-DNA hybridization. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α, adiponectin, leptin, resistin, and vitamin D were measured at baseline. Sample collection was then performed in a subset of the population 6 months after therapy (n = 17). Serum samples were analyzed using enzyme-linked immunosorbent assay and immunoassays. Differences in clinical, microbial, and serum factors among groups were sought using the Mann-Whitney U test. Correlations among factors were evaluated using regression analysis. Effects of therapy were sought using the Wilcoxon signed rank test. RESULTS: There were positive correlations between adiponectin/vitamin D and between IL-6/leptin, negative correlations between IL-6/vitamin D and leptin/vitamin D, but no associations between serum analytes and clinical or microbial parameters. Sex and body mass index were associated with levels of adipokines. Periodontal therapy improved clinical and microbiologic parameters but did not influence the levels of serum analytes. CONCLUSION: Adipokines and IL-6 levels were affected by sex and body mass index. Serum analytes were not influenced by periodontal therapy.

Microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease.

AIM: To monitor microbial shifts during dental biofilm re-development. MATERIALS AND METHODS: Supra- and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects at baseline and immediately after tooth cleaning. Samples were taken again from seven teeth in randomly selected quadrants during 1, 2, 4 and 7 days of no oral hygiene. Samples were analysed using checkerboard DNA-DNA hybridization. Species counts were averaged within subjects at each time point. Significant differences in the counts between healthy and periodontitis subjects were determined using the Mann-Whitney test. RESULTS: The total supra- and subgingival counts were significantly higher in periodontitis on entry and reached or exceeded the baseline values after day 2. Supragingival counts of Veillonella parvula, Fusobacterium nucleatum ss vincentii and Neisseria mucosa increased from 2 to 7 days. Subgingival counts were greater for Actinomyces, green and orange complex species. Significant differences between groups in supragingival counts occurred for 17 of 41 species at entry, 0 at day 7; for subgingival plaque, these values were 39/41 taxa at entry, 17/41 at day 7. CONCLUSIONS: Supragingival plaque re-development was similar in periodontitis and health, but subgingival species recolonization was more marked in periodontitis.

Relationships between subgingival microbiota and GCF biomarkers in generalized aggressive periodontitis.

AIM: To examine relationships between subgingival biofilm composition and levels of gingival crevicular fluid (GCF) cytokines in periodontal health and generalized aggressive periodontitis (GAP). MATERIALS AND METHODS: Periodontal parameters were measured in 25 periodontally healthy and 31 GAP subjects. Subgingival plaque and GCF samples were obtained from 14 sites from each subject. Forty subgingival taxa were quantified using checkerboard DNA-DNA hybridization and the concentrations of eight GCF cytokines were measured using Luminex. Cluster analysis was used to define sites with similar subgingival microbiotas in each clinical group. Significance of differences in clinical, microbiological and immunological parameters among clusters was determined using the Kruskal-Wallis test. RESULTS: GAP subjects had statistically significantly higher GCF levels of interleukin-1beta (IL-1beta) (p<0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (p<0.01) and IL-1beta/IL-10 ratio (p<0.001) and higher proportions of Red and Orange complex species than periodontally healthy subjects. There were no statistically significant differences in the mean proportion of cytokines among clusters in the periodontally healthy subjects, while the ratio IL-1beta/IL-10 (p<0.05) differed significantly among clusters in the aggressive periodontitis group. CONCLUSIONS: Different subgingival biofilm profiles are associated with distinct patterns of GCF cytokine expression. Aggressive periodontitis subjects were characterized by a higher IL-1beta/IL-10 ratio than periodontally healthy subjects, suggesting an imbalance between pro- and anti-inflammatory cytokines in aggressive periodontitis.

The reproducibility of curet sampling of subgingival biofilms.

BACKGROUND: Because few studies have examined the critical issue of sampling reproducibility, the purpose of the present study was to examine the reproducibility of curet sampling of subgingival biofilms. METHODS: Seven subgingival biofilm samples were taken successively, using a curet, from each of 80 sites and individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. One healthy site was sampled in each of 20 periodontally healthy subjects, and one sulcus/pocket of < or =3, 4 to 5, and > or =6 mm was sampled in each of 20 subjects with chronic periodontitis. The significance of differences in counts and proportions of individual species at the seven successive samplings for each probing depth (PD) category was determined using the Kruskal-Wallis test. The reproducibility of species proportions at each PD category was measured using the coefficient of variation (CV), and the consistency of microbial profiles across samples was examined using the minimum similarity coefficient. RESULTS: There was no significant difference in the mean proportions of the 40 test species in the seven successive samples in each of the four PD categories. The median CV for individual species in the same site was 0.79 (95% confidence interval [CI]: 0.76 to 0.82) compared to 1.76 (95% CI: 1.69 to 1.82) in samples from different sites. The within-site mean minimum similarity coefficient (+/- SEM) was 51.2% +/- 2.2%, and it was 27.9% +/- 0.3% between sites. CONCLUSION: The proportions of species remained consistent in successive curet samples, indicating that the use of curets provided a reliable and reproducible method to obtain subgingival samples.