F. Elizabeth Martin

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 Bergeys 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 Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

ABSTRACT 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.

Molecular Analysis of Microbial Diversity in Advanced Caries

ABSTRACT 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.

Carious Dentine Provides a Habitat for a Complex Array of Novel Prevotella-Like Bacteria

ABSTRACT 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.

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 manufacturers 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.

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.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.

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.