RNA-Oligonucleotide Quantification Technique (ROQT) for the Enumeration of Uncultivated Bacterial Species in Subgingival Biofilms

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 104 cells. Fusobacterium nucleatum ss polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples

Relationships Among IL-6, TNF-α, Adipokines, Vitamin D and Chronic Periodontitis

Objectives to explore relationships among serum adipokines, vitamin D, 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 (BOP), suppuration, pocket depth (PD) and attachment level (AL) 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 (TNF-α), adiponectin, leptin, resistin and vitamin D were measured at baseline. Sample collection was then performed in a subset of the population 6 months post-therapy (n=17). Serum samples were analyzed using ELISA and immunoassays. Differences in clinical, microbial and serum factors among groups were sought using the Mann-Whitney test. Correlations among factors were evaluated using regression analysis. Effects of therapy were sought using the Wilcoxon signed ranks 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. Gender and BMI were associated with levels of adipokines. Periodontal therapy improved clinical and microbiological parameters, but did not influence the levels of serum analytes. Conclusions Adipokines and IL-6 levels were affected by gender and BMI. Serum analytes were not influenced by periodontal therapy

Early Microbial Succession in Re-Developing Dental Biofilms in Periodontal Health and Disease

Objective To determine the order of bacterial species succession in re-developing supra and subgingival biofilms. Methods Supra 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 7 teeth in randomly selected quadrants after 1, 2, 4 and 7 days of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. % 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 periodontitis and health was similar. At 1 day, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 days. At 4–7 days, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 days by Capnocytophaga sputigena and P. nigrescens. No significant increase in proportions of periodontal pathogens was observed in any of the clinical groups or locations. Conclusions There is a defined order in bacterial species succession in early supra and subgingival biofilm re-development after professional cleaning

Comparison of Microbial Changes in Early Re-Developing Biofilms on Natural Teeth and Dentures

Background and objective Surfaces and fluids can affect oral bacterial colonization. The aim of this study was to compare re-developing biofilms on natural teeth and dentures. Methods Supragingival plaque samples were taken from 55 dentate subjects and the denture teeth of 62 edentulous subjects before and after professional cleaning. Also, samples from 7 “teeth” 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 sought using the Mann-Whitney test. Ecological succession was determined using a modified moving window analysis. Results Mean total DNA probe counts were similar pre-cleaning but were higher in dentate subjects at all post-cleaning visits (pStreptococcus mitis, Streptococcus oralisand Streptococcus mutans, whereas dentate subjects had higher proportions of Tannerella forsythia, Selenomonas noxia and Neisseria mucosa. By 2 days, mean counts of all taxa were higher in natural teeth and most remained higher at 7 days (pS. mitis and S. oralis by 1 day. N. mucosa, Veillonella parvula and Eikenella corrodens increased in both groups but later in edentate samples. Conclusions “Mature” natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm re-development is more rapid and more complex on natural than denture teeth

Effects of Azithromycin, Metronidazole, Amoxicillin, and Metronidazole plus Amoxicillin on an In Vitro Polymicrobial Subgingival Biofilm Model

ABSTRACT Chronic periodontitis is one of the most prevalent human diseases and is caused by dysbiosis of the subgingival microbiota. Treatment involves primarily mechanical disruption of subgingival biofilms and, in certain cases, adjunctive use of systemic antibiotic therapy. In vitro biofilm models have been developed to study antimicrobial agents targeting subgingival species. However, these models accommodate a limited number of taxa, lack reproducibility, and have low throughput. We aimed to develop an in vitro multispecies biofilm model that mimics subgingival plaque, to test antimicrobial agents. Biofilms were cultivated using the Calgary Biofilm Device and were exposed to amoxicillin (AMX), metronidazole (MTZ), azithromycin (AZM), and AMX-MTZ at four different concentrations for 12, 24, or 36 h. Chlorhexidine (CHX) (0.12%) was used as the positive control. The compositions of the biofilms were analyzed by checkerboard DNA-DNA hybridization, and the percent reduction in biofilm metabolic activity was determined using 2,3,5-triphenyltetrazolium chloride and spectrophotometry. Thirty-five of the 40 species used in the inoculum were consistently recovered from the resulting in vitro biofilms. After 36 h of exposure at the 1:27 dilution, AMX-MTZ reduced metabolic activity 11% less than CHX ( q = 0.0207) but 54% more than AMX ( q = 0.0031), 72% more than MTZ ( q = 0.0031), and 67% more than AZM ( q = 0.0008). Preliminary evidence of a synergistic interaction between AMX and MTZ was also observed. In summary, we developed reproducible biofilms with 35 subgingival bacterial species, and our results suggested that the combination of AMX and MTZ had greater antimicrobial effects on these in vitro multispecies biofilms than expected on the basis of the independent effects of the drugs

Development and validation of HERWIG 7 tunes from CMS underlying-event measurements

This paper presents new sets of parameters (“tunes”) for the underlying-event model of the HERWIG7 event generator. These parameters control the description of multiple-parton interactions (MPI) and colour reconnection in HERWIG7, and are obtained from a fit to minimum-bias data collected by the CMS experiment at s=0.9, 7, and 13Te. The tunes are based on the NNPDF 3.1 next-to-next-to-leading-order parton distribution function (PDF) set for the parton shower, and either a leading-order or next-to-next-to-leading-order PDF set for the simulation of MPI and the beam remnants. Predictions utilizing the tunes are produced for event shape observables in electron-positron collisions, and for minimum-bias, inclusive jet, top quark pair, and Z and W boson events in proton-proton collisions, and are compared with data. Each of the new tunes describes the data at a reasonable level, and the tunes using a leading-order PDF for the simulation of MPI provide the best description of the dat

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe