Development of the oral resistome during the first decade of life.

Antibiotic overuse has promoted the spread of antimicrobial resistance (AMR) with significant health and economic consequences. Genome sequencing reveals the widespread presence of antimicrobial resistance genes (ARGs) in diverse microbial environments. Hence, surveillance of resistance reservoirs, like the rarely explored oral microbiome, is necessary to combat AMR. Here, we characterise the development of the paediatric oral resistome and investigate its role in dental caries in 221 twin children (124 females and 97 males) sampled at three time points over the first decade of life. From 530 oral metagenomes, we identify 309 ARGs, which significantly cluster by age, with host genetic effects detected from infancy onwards. Our results suggest potential mobilisation of ARGs increases with age as the AMR associated mobile genetic element, Tn916 transposase was co-located with more species and ARGs in older children. We find a depletion of ARGs and species in dental caries compared to health. This trend reverses in restored teeth. Here we show the paediatric oral resistome is an inherent and dynamic component of the oral microbiome, with a potential role in transmission of AMR and dysbiosis.

Influence of implant abutment angulations and two types of fibers on the fracture resistance of ceramage single crowns.

PURPOSE: To assess the effect of three implant abutment angulations and two types of fibers on the fracture resistance of overlaying Ceramage single crowns. MATERIALS AND METHODS: Three groups, coded A to C, with different implant abutment angulations (group A/0°, group B/15°, and group C/30° angulation) were restored with 45 overlay composite restorations; 15 Ceramage crowns for each angulation. Groups A, B, and C were further subdivided into three subgroups (n = 5) coded: 1, crowns without fiber reinforcement; 2, crowns with Connect polyethylene reinforcement; and 3, crowns with Interlig glass reinforcement. All crowns were constructed by one technician using the Ceramage System. The definitive restorations (before cementation) were stored in distilled water at mouth temperature (37°C) for 24 hours prior to testing. Before testing, the crowns were cemented using Temp Bond. The compressive load required to break each crown and the mode of failure were recorded. The speed of testing was 1 mm/min. The results were statistically analyzed by two-way ANOVA (p < 0.05). The tested crowns were examined using a stereomicroscope at 40×, and selected crowns (five randomly selected from each group) were further examined by scanning electron microscopy (SEM) to reveal the composite-fiber interface. RESULTS: Fracture resistance of single crowns was not affected (p > 0.05) by the different abutment angulations chosen (0°, 15°, 30°) or fiber reinforcement (Connect and Interlig fibers). Crowns in group A exhibited average loads to fracture (N) of A1 = 843.57 ± 168.20, A2 = 1389.20 ± 193.40, and A3 = 968.00 ± 387.53, which were not significantly different (p > 0.05) from those of groups B (B1 = 993.20 ± 327.19, B2 = 1471.00 ± 311.68, B3 = 1408.40 ± 295.07), or group C (C1 = 1326.80 ± 785.30, C2 = 1322.20 ± 285.33, C3 = 1348.40 ± 527.21). SEM images of the fractured crowns showed that the origin of the fracture appeared to be located at the occlusal surfaces of the crowns, and the crack propagation tended to extend from the occlusal surface towards the gingival margin. CONCLUSIONS: Implant abutment angulations of 0°, 15°, and 30° did not significantly (p > 0.05) influence the fracture resistance of overlaying Ceramage single crowns constructed with or without reinforcing fibers. The two types of fibers used for reinforcement (Connect and Interlig) had no effect (p > 0.05) on the fracture resistance of overlaying Ceramage single crowns.

Quantifying the selection of maxillary anterior teeth using intraoral and extraoral anatomical landmarks.

BACKGROUND: One of the major hurdles in clinical prosthodontics has been the selection and replacement of maxillary anterior teeth in the absence of pre-extraction records. The aim of this study was to determine if a relationship exists between intraoral and extraoral facial measurements that could assist dental practitioners in selecting esthetically appropriate maxillary anterior teeth in the absence of pre-extraction records. MATERIALS AND METHODS: A cross-sectional study design was used with a sample size of one hundred and twenty participants. A questionnaire was used to identify the selection criteria and a photograph was taken for facial measurements using digitally calibrated software. Ninety-eight participants met the selection criteria and were included in the study. Measurements of intraoral landmarks were taken from stone casts of maxillary impressions using calibrated digital calipers. Each measurement was completed by two assessors to obtain mean values. Data were statistically analyzed using SPSS version 17 software. Data were assessed by one way analysis of variance (ANOVA) followed by post hoc (p < 0.05) to find any difference between tested groups. Pearson coefficients were used to determine whether correlation exists between measurements. RESULTS: The mean values for intraoral maxillary landmarks were: Central incisor width = 8.39 mm, circumferential canine tip to canine tip distance = 34.89 mm, arch width = 48.24 mm, left arch length = 45.24 mm, right arch length = 45.56 mm. The mean values for extraoral landmarks were: Intercanthal distance = 33.24 mm, interpupillary distance = 60.68 mm, interalar distance = 38.27 mm, intercommissure distance = 50.61 mm. Differences existed within subgroups for all intraoral and extraoral measures. A weak positive correlation existed between intraoral (r < 0.4) and extraoral measurements (r < 0.38) that remained consistent when examined by gender. CONCLUSION: This study showed that the average length and width of the maxillary arch and interalar width were the anatomical landmarks that provided the strongest predictive relationship with anterior maxillary teeth (r = 0.38 - 0.4). Using these dimensions an average multiplying factor can be used to calculate maxillary incisor width or canine tip to canine tip distance. As the predictive strength is not strong, the authors recommend its use as a preliminary guide for determining the width of the maxillary anterior teeth during the initial selection of artificial teeth in the absence of pre-extraction records. CLINICAL SIGNIFICANCE: The results of this study can be used to help dentists select the size of artificial maxillary anterior teeth in the absence of pre-extraction records.

Enhancing Fluoride Mediated Dentine Sensitivity Relief through Functionalised Tricalcium Phosphate Activity.

Background. To assess the clinical efficacy of a dentifrice containing fluoride and functionalised tricalcium phosphate (fTCP) in reducing dentine sensitivity. Methods. A 10-week parallel blind randomised control trial was conducted. Subjects were assigned to one of four groups and instructed to brush twice daily: A: Colgate Cavity Protection (1000 ppmF-MFP); B: Sensodyne Total Care (1000 ppmF-NaF + 19300 ppmK(+)-KNO3); C: Clinpro Tooth Crème (950 ppmF-NaF + fTCP); and D: Clinpro Tooth Crème (brushing + additional topical application). Seventy-one patients were assessed at baseline, 6 weeks, and 10 weeks for cold, tactile, and hypertonic sensitivity using the NRS-11 pain rating scale. A combined modalities sensitivity score (CMS) was calculated. Results. At 6 weeks, patients reported the following reduction in CMS: A (20%); B (30%); C (42%); D (52%). At 10 weeks, patients reported the following reduction in CMS: A (18%), B (40%), C (24%), and D (54%). The only CMS comparisons to show a significant difference (P < 0.05) were between Groups A and D (6 and 10 weeks). Conclusions. Addition of fTCP to a dentifrice enhances the ability of dentifrice fluoride in reducing dentine sensitivity. Using Clinpro Tooth Crème twice daily for brushing can be as effective to reduce dentine sensitivity as twice daily brushing using Sensodyne Total Care. However, additional nightly topical application of fTCP, in addition to twice daily brushing, showed an enhanced reduction in dentine sensitivity.

Carious pulpitis: microbiological and histopathological considerations.

Dental caries is the result of microbial activities that induce the progressive localised destruction of teeth. Without treatment, this eventually results in infection of the dental pulp and surrounding periapical tissues. Although the bacteria responsible for caries initiation and early caries progression have been extensively studied, the microbiology of dentine caries reportedly shows considerable diversity and the associated microflora has not yet been fully identified. A search of the literature shows that few studies have analysed the microbiology of deep caries or examined the relationship between this microflora and the histopathology of chronic pulpitis in symptomatic teeth. The majority of the studies investigating the microbiology of carious dentine have used traditional culture methodology that has been reported to be fraught with difficulties and to underestimate the microbial populations. However, recent work using new technology in the form of Polymerase Chain Reaction (PCR) has shown potential by enhancing the identification and quantification of bacteria from complex environments. Application of this technology to carious dentine has identified an environment dominated by anaerobic organisms and containing significant numbers of Gram-negative bacteria that have been strongly implicated in endodontic infections subsequent to carious pulpitis. Examination of the histopathology of pulp sections from teeth extracted as a result of carious pulpitis showed pulpal reactions ranging from minimal inflammation to marked inflammatory infiltration of the pulp tissue. Of interest, were hard and soft tissue pathologic changes noted in the pulp tissues resulting from the combined effects of the carious microorganisms and the host tissue response. Improved knowledge of the microbial species associated with pulpitis could create the potential for development of diagnostic tools and restorative materials with appropriate antimicrobial properties.

Influence of immediate dentin sealing on the shear bond strength of pressed ceramic luted to dentin with self-etch resin cement.

Objectives. To examine the effect of immediate dentin sealing (IDS), with dentin bonding agents (DBAs) applied to freshly cut dentin, on the shear bond strength of etched pressed ceramic luted to dentin with RelyX Unicem (RXU) cement. Method. Eighty extracted noncarious third molars were ground flat to expose the occlusal dentin surfaces. The teeth were randomly allocated to five groups (A to E) of sixteen teeth each. Groups A to D were allocated a dentin bonding agent (Optibond FL, One Coat Bond, Single Bond, or Go!) that was applied to the dentin surface to mimic the clinical procedure of IDS. These specimen groups then had etched glass ceramic discs (Authentic) luted to the sealed dentin surface using RXU. Group E (control) had etched glass ceramic discs luted to the dentin surface (without a dentin bonding agent) using RXU following the manufacturer's instructions. All specimens were stored for one week in distilled water at room temperature and then shear stressed at a constant cross-head speed of 1 mm per minute until failure. Statistical analysis was performed by ANOVA followed by post hoc Tukey HSD method (P < 0.05) applied for multiple paired comparisons. Results. The shear bond strength results for group A to E ranged from 6.94 ± 1.53 to 10.03 ± 3.50 MPa. One-way ANOVA demonstrated a difference (P < 0.05) between the groups tested and the Tukey HSD demonstrated a significant (P < 0.05) difference between the shear bond strength (SBS) of Optibond FL (Group A) and Go! (Group D). There was no statistical difference (P > 0.05) in the SBS between the test groups (A-D) or the control (group E). Conclusion. IDS using the dentin bonding agents tested does not statistically (P > 0.05) affect the shear bond strength of etched pressed ceramic luted to dentin with RXU when compared to the control.

Polymerization profile analysis of resin composite dental restorative materials in real time.

OBJECTIVES: In this study, the hypothesis that the polymerization shrinkage profile of "low shrinkage" non-methacrylate based composite; Silorane and "low shrinkage" high molecular mass methacrylate based composite; Kalore is not different from that of three conventional methacrylate based composites (Gradia Direct X, Filtek Supreme XT and Beautifil II) was tested. METHODS: Five commercially available composites were analysed: one "low shrinkage" non-methacrylate based composite (Silorane); one "low shrinkage" high molecular mass methacrylate based composite (Kalore) and three conventional methacrylate based composites (Gradia Direct X, Filtek Supreme XT and Beautifil II). Polymerization shrinkage was measured using an electromagnetic balance which recorded changes in composite buoyancy occurring due to volumetric changes during polymerization. This instrument allowed real time volumetric shrinkage measurements to be made at 40 ms intervals. RESULTS: All five resin composites demonstrated a similar volumetric shrinkage profile during polymerization. The rate of shrinkage of all five composites decreased from t=0 at a rate approximating x=t. After 170 s the rate of shrinkage of all five composites was at or below 0.01%/s. During the initial 5s of light exposure Silorane and Kalore exhibited a significantly lower (p<0.05) rate of contraction relative to the three conventional methacrylate composites. After 640 s of analysis, Silorane exhibited a significantly lower (p<0.05) percentage volumetric contraction compared to the other four analysed materials. CONCLUSIONS: The newly developed "low shrinkage" composites (Silorane, Kalore) in the present study demonstrated significantly lower (p<0.05) shrinkage rates and shrinkage volumes compared to the three conventional methacrylate composites. Investigation to identify whether polymerization shrinkage profile analysis is a good predictor of relative polymerization contraction stress levels generated by different composites, is warranted. CLINICAL SIGNIFICANCE: Clinicians making a resin composite selection with the view to minimizing the clinical effects of polymerization shrinkage must consider the rate of polymerization as well as the total volumetric shrinkage of a composite. Silorane (non methacrylate composite) and Kalore (high molecular mass methacrylate composite) have the ability to exhibit lower shrinkage rates and lower shrinkage volumes compared to conventional methacrylate composites.

Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR.

Methods for the optimal extraction of genomic DNA for real-time PCR enumeration of oral bacteria using the muramidase, mutanolysin, were developed using a simple in vitro oral flora model comprised of the facultative anaerobic gram-positive bacteria, Lactobacillus acidophilus and Streptococcus mutans, the gram-positive anaerobe, Parvimonas micra, and the gram-negative anaerobes, Porphyromonas gingivalis, Prevotella melaninogenica and Fusobacterium nucleatum. Traditional, as well as more elaborate, methods of quantifying bacterial numbers, including colony counting and estimation of DNA content using 4',6-diamino-2-phenylindole were compared in order to validate the real-time PCR approach. Evidence was obtained that P. gingivalis nuclease activity adversely affected the extraction of double-stranded DNA from this bacterium either alone or when it formed part of a consortium with the other bacteria. This nuclease activity could be overcome by treatment of the bacteria with either 20 mM diethyl pyrocarbonate or 70% ethanol at 4 degrees C overnight. A final purification of the DNA to remove any potential PCR inhibitors was added to the protocol in order to accurately quantify the amount of DNA by real-time PCR and hence the number of bacteria in a sample.

Adhesive bonding--some clinical considerations.

Over the last decade the development of adhesives has moved from the classic concept of three-step bonding towards the introduction of more user-friendly, simplified systems. Current adhesive systems can be grouped into three categories: etch and rinse adhesives, self-etch materials and glass ionomers. Published data indicate that the three-step etch and rinse systems form the gold standard in durability, with the two-step self-etch materials approaching that standard, and the glass ionomers, although clinically reliable, are limited by their physical properties. However, a major shortcoming of bonded restorations is their unreliable durability in vivo. One of the most important factors influencing bond durability is hydrolysis, which affects all adhesive systems. Two important aspects of this process involve water uptake in the adhesive resin and thinning or disappearance of collagen fibrils from within the hybrid zone. Techniques to improve the stability of the adhesive bond and prolong the clinical life of adhesive restorations are discussed.

Molecular analysis of microbial diversity in advanced caries.

Real-time PCR analysis of the total bacterial load in advanced carious lesions has shown that the total load exceeds the number of cultivable bacteria. This suggests that an unresolved complexity exists in bacteria associated with advanced caries. In this report, the profile of the microflora of carious dentine was explored by using DNA extracted from 10 lesions selected on the basis of comparable total microbial load and on the relative abundance of Prevotella spp. Using universal primers for the 16S rRNA gene, PCR amplicons were cloned, and approximately 100 transformants were processed for each lesion. Phylogenetic analysis of 942 edited sequences demonstrated the presence of 75 species or phylotypes in the 10 carious lesions. Up to 31 taxa were represented in each sample. A diverse array of lactobacilli were found to comprise 50% of the species, with prevotellae also abundant, comprising 15% of the species. Other taxa present in a number of lesions or occurring with high abundance included Selenomonas spp., Dialister spp., Fusobacterium nucleatum, Eubacterium spp., members of the Lachnospiraceae family, Olsenella spp., Bifidobacterium spp., Propionibacterium sp., and Pseudoramibacter alactolyticus. The mechanisms by which such diverse patterns of bacteria extend carious lesions, including the aspect of infection of the vital dental pulp, remain unclear.

Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis.

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

Quantitative analysis of diverse Lactobacillus species present in advanced dental caries.

Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion. Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix. The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR. Pooled DNA was amplified by PCR with Lactobacillus genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis. Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes. Specific PCR primers were designed for nine prominent species, including Lactobacillus gasseri, L. ultunensis, L. salivarius, L. rhamnosus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, and L. gallinarum. More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various Lactobacillus spp. in carious dentine. Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of L. gasseri and L. ultunensis than of the other prevalent species. The findings provide a basis for further characterization of the pathogenicity of Lactobacillus spp. in the context of extension of the carious lesion.

Carious dentine provides a habitat for a complex array of novel Prevotella-like bacteria.

Previous analysis of the microbiology of advanced caries by culture and real-time PCR emphasized the high incidence and abundance of gram-negative anaerobic species, particularly Prevotella-like bacteria. The diversity of Prevotella-like bacteria was further explored by analyzing pooled bacterial DNA from lesions of carious dentine. This was achieved by amplification of a region of the 16S ribosomal DNA with a Prevotella genus-specific forward primer and a universal bacterial reverse primer, followed by cloning and sequencing. Cultured Prevotella species commonly associated with oral tissues constituted only 12% of the Prevotella clones isolated from advanced carious lesions. The remaining 88% consisted of a diverse range of phylotypes. These included five clusters of previously recognized but uncultured oral Prevotella spp. and a major cluster containing Prevotella-like bacteria most closely related to uncharacterized rumen bacteria. Cluster-specific primers were designed, and the numbers of bacteria within clusters were quantified by real-time PCR, confirming the abundance of these organisms. The data indicated that advanced dental caries provides a unique environment for a complex array of novel and uncultured Prevotella and Prevotella-like bacteria which, in some cases, may dominate the diverse polymicrobial community associated with the disease.