Glycaemic status affects the subgingival microbiome of diabetic patients.

AIM: Periodontitis is correlated with type 2 diabetes mellitus (T2DM), but little is known about glycaemic status effect on subgingival microbiota associated with periodontitis. This study evaluated if periodontal microbiome of T2DM patients is affected by glycaemic status. MATERIALS AND METHODS: Twenty-one T2DM non-smoking patients with chronic periodontitis and body mass index ≤40 kg/m2 were allocated into two groups according to systemic glycaemic status: inadequate (DMI- HbA1c ≥ 8%) and adequate (DMA- HbA1c <7.8%). Subgingival biofilm was collected from sites with moderate (PD = 4-6 mm) and severe disease (PD ≥ 7 mm) in two quadrants. The V5-V6 hypervariable region of the 16SrRNA was sequenced using the GS-FLX-454 Titanium platform. Sequences were compared with HOMD database using QIIME and PhyloToAST pipelines. Statistical comparisons were made using two-sample t-tests. RESULTS: DMA microbiome presented higher diversity than DMI. Inadequate glycaemic control favoured fermenting species, especially those associated with propionate/succinate production, whereas those forming butyrate/pyruvate was decreased in DMI. Higher abundances of anginosus group and Streptococcus agalactiae in DMI may indicate that subgingival sites can be reservoir of potentially invasive pathogens. Altered subgingival microbiome in DMI may represent an additional challenge in the periodontal treatment of these patients and in the prevention of more invasive infections. CONCLUSION: Glycaemic status in T2DM patients seems to modulate subgingival biofilm composition.

Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable Enterococcus faecalis.

INTRODUCTION: One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis. METHODS: Viable or heat-killed suspensions of E. faecalis (106 colony-forming units/mL) were treated with PMA at 10, 50, and 100 µg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls. RESULTS: The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 µg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells. CONCLUSIONS: PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.