Impact of estradiol and quorum sensing on biofilm-related virulence of the Prevotella intermedia group: – in vitro studies on bacterial growth properties, proteolytic enzyme activity and cytokine production

The Prevotella intermedia group organisms (P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca) are Gram-negative anaerobes predominantly existing in the human oral cavity. P. intermedia and P. nigrescens are characterized by their ability to consume maternal steroids; estrogen and progesterone. During pregnancy, microbial shifts in subgingival microbiota favoring P. intermedia, the tendency for gingival bleeding and elevated serum levels of maternal steroids occur. P. intermedia sensu lato (P. intermedia and P. nigrescens) is able to utilize estrogen and progesterone as a nutritional source, thus, it is here hypothesized that estradiol, the most potent estrogen during pregnancy, has an impact on the growth properties and pathogenicity of the P. intermedia group organisms. The aim of this study series was to examine the effect of estradiol on the growth behavior, biofilm formation and proteolytic enzyme activity of the P. intermedia group bacteria. Secondly, the impact of estradiol and QS on gingival keratinocyte response against the cell extracts of P. intermedia group bacteria, was determined based on the cytokine response. Thirdly, the role of quorum sensing (QS) molecules as enzyme and biofilm inhibitors was evaluated. The nine strains of P. intermedia group bacteria used in the study were: P. intermedia ATCC 25611T and AHN 8290, P. nigrescens ATCC 33563T and AHN 8293, P. pallens NCTC 13042T and AHN 9283, and P. aurantiaca AHN 37505, AHN 37552, and CCUG 57723. In addition, Fusobacterium nucleatum ATCC 25586T was used in the coaggregation assays. Colony forming unit, spectrophotometric, Bradford and phenol-sulfuric acid methods were used to examine the planktonic growth, coaggregation, biofilm formation, and protein and polysaccharide levels, respectively. Enzyme activities of the P. intermedia group bacteria were determined with fluorometric methods and zymography, while cytokine responses were analyzed by Luminex multiplex immunoassay. The study indicated that estradiol has a significant regulatory effect on the biofilm-related virulence of P. intermedia group organisms in a dose- and strain-dependent manner. QS molecules may be considered a promising therapeutic target in the treatment of oral chronic conditions related to bacterial biofilms, including pregnancy-associated gingivitis

Prevotella species as oral residents and infectious agents with potential impact on systemic conditions

Oral Prevotella are known as anaerobic commensals on oral mucosae and in dental plaques from early life onwards, including pigmented P. melaninogenica, P. nigrescens, and P. pallens and non-pigmented Prevotella species. Many Prevotella species contribute to oral inflammatory processes, being frequent findings in dysbiotic biofilms of periodontal diseases (P. intermedia, P. nigrescens), cariotic lesions (P. denticola, Alloprevotella (formerly Prevotella) tannerae), endodontic infections (P. baroniae, P. oris, P. multisaccharivorax), and other clinically relevant oral conditions. Over the years, several novel species have been recovered from the oral cavity without knowledge of their clinical relevance. Within this wide genus, virulence properties and other characteristics like biofilm formation seemingly vary in a species- and strain-dependent manner, as shown for the P. intermedia group organisms (P. aurantiaca, P. intermedia, P. nigrescens, and P. pallens). Oral Prevotella species are identified in various non-oral infections and chronic pathological conditions. Here, we have updated the knowledge of the genus Prevotella and the role of Prevotella species as residents and infectious agents of the oral cavity, as well as their detection in non-oral infections, but also gathered information on their potential link to cancers of the head and neck, and other systemic disorders.</p

Elevated baseline salivary protease activity may predict the steadiness of gingival inflammation during periodontal healing:a 12-week follow-up study on adults

© 2020 by the authors. Licensee MDPI, Basel, Switzerland.Aim was to profile salivary total protease, Porphyromonas gingivalis gingipain, and neutrophil elastase activities in relation to the resolution of periodontal inflammation, salivary macrophage-derived chemokine (MDC), and macrophage inflammatory protein-1α concentrations. Nonsurgical periodontal treatment was performed in 24 periodontitis patients in a prospective interventional study design. Periodontal clinical parameters were recorded, and stimulated saliva samples were collected at baseline and 2, 6, and 12 weeks after treatment. Salivary total protease and gingipain activities were determined using fluorogenic substrates, elastase activity by chromogenic substrates, and cytokine concentrations by Luminex immunoassay. For statistical analyses, generalized linear mixed models for repeated measures were used. Salivary total protease activity elevated, while gingival inflammation and plaque accumulation decreased 2 and 6 weeks after periodontal therapy. Salivary MDC concentration was elevated 12 weeks after periodontal treatment. Patients with elevated protease activities at baseline in comparison to patients with low baseline total protease activities, had higher levels of gingival inflammation before and after periodontal treatment. In conclusion, elevations in salivary total protease activity seem to be part of periodontal healing at its early phases. Higher levels of salivary total protease activities before periodontal treatment may predict the severity and steadiness of unresolved gingival inflammation

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes

Dental implant material has an impact on adhesion and spreading of oral mucosal cells on its surface. Platelet-rich fibrin (PRF), a second-generation platelet concentrate, can enhance cell proliferation and adhesion. The aim was to examine the regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes. Human gingival keratinocytes were cultured on titanium grade 4, titanium grade 5 (Ti5), and HA discs at 37 °C in a CO2 incubator for 6 h and 24 h, using either elutes of titanium-PRF (T-PRF) or leukocyte and platelet-rich fibrin (L-PRF), or mammalian cell culture medium as growth media. Cell numbers were determined using a Cell Titer 96 assay. Interleukin (IL)-1β, IL-1Ra, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) expression levels were measured using the Luminex® xMAP™ technique, and cell adhesion and spread by scanning electron microscopy. Epithelial cell adhesion and spread was most prominent to Ti5 surfaces. L-PRF stimulated cell adhesion to HA surface. Both T-PRF and L-PRF activated the expressions of IL-1 β, IL-8, IL-1Ra, MCP-1, and VEGF, T-PRF being the strongest activator. Titanium surface type has a regulatory role in epithelial cell adhesion and spread, while PRF type determines the cytokine response.</p

Gingival tissue human beta-defensin levels in relation to infection and inflammation.

Aim To profile gingival tissue levels of human beta-defensin (hBD)-2 and hBD-3 in relation to gingival inflammation, Th17-related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities. Materials and Methods Gingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real-time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation coefficients. Results Subgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p <.001), as well as interleukin (IL)-1 beta (p = .012), IL-10 (p = .024), IL-17A (p = .002), IL-17F (p = .006), and IL-23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD-2 and hBD-3 levels. Negative correlations were found between protease activity and hBD-2 (p = .033) and hBD-3(p = .003) levels. Conclusions Shift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.Peer reviewe

Gingival Tissue Human Beta-Defensin Levels in Relation to Infection and Inflammation

AimTo profile gingival tissue levels of human beta‐defensin (hBD)‐2 and hBD‐3 in relation to gingival inflammation, Th17‐related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities.Materials and MethodsGingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real‐time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal–Wallis and Mann–Whitney U tests and Spearman correlation coefficients.ResultsSubgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p p = .012), IL‐10 (p = .024), IL‐17A (p = .002), IL‐17F (p = .006), and IL‐23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD‐2 and hBD‐3 levels. Negative correlations were found between protease activity and hBD‐2 (p = .033) and hBD‐3(p = .003) levels.ConclusionsShift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.</p

Ultrastructural changes of Fusobacterium nucleatum as a defense mechanism against human neutrophil peptide-1 - A Transmission Electron Microscopy (TEM) study

Aims: The aim of this work was to assess the ultrastructural changes, cellular proliferation, and the biofilm formation ability of F. nucleatum as defense mechanisms against the effect of HNP-1. Materials and methods: The type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were cultured and incubated with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml) and one control group (0 µg/ml). Bacterial pellets from each concentration were processed for TEM imaging. Planktonic growth was assessed and colony forming units (CFU) were measured to determine the cellular proliferation. Scrambled HNP-1 was used for confirmation. Results: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. For ATCC 25586, the treated bacterial cells contained higher numbers of intracellular granules with increasing the peptide concentration. Planktonic growth of the two clinical strains were significantly enhanced (P<0.001) with gradually increased concentrations of HNP-1. None of the planktonic growth results of the 3 strains incubated with the scrambled HNP-1 was statistically significant. HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 and 9508, significantly (P<0.01 and P<0.001; respectively). Conclusions: The present in vitro study demonstrates that F. nucleatum has the ability to withstand the lethal effects of HNP-1 even at concentrations simulating the diseased periodontium in vivo. The increase in planktonic growth could act as defense mechanisms of F. nucleatum against HNP-1.Siirretty Doriast

Comparison of physical quality assurance between Scanora 3D and 3D Accuitomo 80 dental CT scanners

Background: The use of cone beam computed tomography (CBCT) in dentistry has proven to be useful in the diagnosis and treatment planning of several oral and maxillofacial diseases. The quality of the resulting image is dictated by many factors related to the patient, unit, and operator. Materials and methods: In this work, two dental CBCT units, namely Scanora 3D and 3D Accuitomo 80, were assessed and compared in terms of quantitative effective dose delivered to specific locations in a dosimetry phantom. Resolution and contrast were evaluated in only 3D Accuitomo 80 using special quality assurance phantoms. Results: Scanora 3D, with less radiation time, showed less dosing values compared to 3D Accuitomo 80 (mean 0.33 mSv, SD±0.16 vs. 0.18 mSv, SD±0.1). Using paired t-test, no significant difference was found in Accuitomo two scan sessions (p>0.05), while it was highly significant in Scanora (p>0.05). The modulation transfer function value (at 2 lp/mm), in both measurements, was found to be 4.4%. The contrast assessment of 3D Accuitomo 80 in the two measurements showed few differences, for example, the grayscale values were the same (SD=0) while the noise level was slightly different (SD=0 and 0.67, respectively). Conclusions: The radiation dose values in these two CBCT units are significantly less than those encountered in systemic CT scans. However, the dose seems to be affected more by changing the field of view rather than the voltage or amperage. The low doses were at the expense of the image quality produced, which was still acceptable. Although the spatial resolution and contrast were inferior to the medical images produced in systemic CT units, the present results recommend adopting CBCTs in maxillofacial imaging because of low radiation dose and adequate image quality