John D. Ruby

Treponema denticola activates mitogen-activated protein kinase signal pathways through Toll-like receptor 2.

ABSTRACT Treponema denticola, a spirochete indigenous to the oral cavity, is associated with host inflammatory responses to anaerobic polymicrobial infections of the root canal, periodontium, and alveolar bone. However, the cellular mechanisms responsible for the recognition of T. denticola by the innate immune system and the underlying cell signaling pathways that regulate the inflammatory response to T. denticola are currently unresolved. In this study, we demonstrate that T. denticola induces innate immune responses via the utilization of Toll-like receptor 2 (TLR2) but not TLR4. Assessment of TLR2/1 and TLR2/6 heterodimers revealed that T. denticola predominantly utilizes TLR2/6 for the induction of cellular responses. Analysis of the mitogen-activated protein kinase (MAPK) signaling pathway in T. denticola-stimulated monocytes identified a prolonged up-regulation of the MAPK extracellular signal-related kinase 1/2 (ERK1/2) and p38, while no discernible increase in phospho-c-Jun N-terminal kinase 1/2 (JNK1/2) levels was observed. With the aid of pharmacological inhibitors selectively targeting ERK1/2 via the mitogen-activated protein kinase/extracellular signal-related kinase 1/2 kinase and p38, we further demonstrate that ERK1/2 and p38 play a major role in T. denticola-mediated pro- and anti-inflammatory cytokine production.

Repetitive Extragenic Palindromic PCR for Study of Streptococcus mutans Diversity and Transmission in Human Populations

ABSTRACT Pulsed-field gel electrophoresis (PFGE) is considered the “gold standard” for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.

Observations of biofilm growth on human dentin and potential destruction after exposure to antibiotics

OBJECTIVES This study was performed to observe biofilm formation on dentin and to then observe effects of clinically achievable antimicrobial drug concentrations on these biofilms. STUDY DESIGN Wild-strain endodontic bacteria were anaerobically cultured from necrotic pulps of extracted human teeth and used to grow biofilms on sterilized dentin slices in an anaerobic chamber for 12 days. Then these biofilms were exposed to ampicillin, doxycycline, clindamycin, azithromycin, or metronidazole. Each day for 8 days, specimens were fixed using 2% glutaraldehyde and examined with a scanning electron microscope (SEM). RESULTS The SEM images revealed the presence of a mature biofilm after 8 days of growth and that none of the antibiotics tested was effective in eliminating the biofilm even after 8 days of exposure. CONCLUSION Biofilms are formed in a few days and are resistant to antimicrobial drugs.

Genetic Diversity of Plaque Mutans Streptococci with rep-PCR

Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.

Genetic characterization of the oral Actinomyces.

Actinomyces are difficult to identify using serological and biochemical methods but genotyping is an efficient and reliable means of bacterial characterization and can be used to determine clonal identity. The purpose here was to genotype 13 American type culture collection (ATCC) reference strains representing six different oral Actinomyces spp. by using chromosomal DNA fingerprinting (CDF), arbitrarily primed-polymerase chain reaction (AP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In CDF analysis, BamHI, BstEII and SmaI yielded digestion patterns revealing characteristic differences among the known Actinomyces spp., with SmaI demonstrating optimal resolution. Amplicons generated by AP-PCR with primer OPB-07 displayed banding patterns that permitted discrimination of all Actinomyces strains tested. PCR-RFLP with MnlI digests generated fragment patterns that also characterized the reference strains. Collectively, genotypic profiles generated by CDF, AP-PCR and PCR-RFLP permitted differentiation of all 13 ATCC Actinomyces strains. SmaI CDF analysis of 18 clinical isolates of catalase-positive A. naeslundii genospecies 2 revealed extensive genetic diversity among these strains. These molecular approaches may be useful in determining genetic diversity within oral Actinomyces populations and fidelity of Actinomyces transmission between mother and child.

The Caries Phenomenon: A Timeline from Witchcraft and Superstition to Opinions of the 1500s to Today's Science

This historical treatise follows the documented timeline of tooth decay into todays understanding, treatment, and teaching of caries biology. Caries has been attributed to many different causes for several millennia, however, only since the late 1900s has research revealed its complex multifactorial nature. European writers of the 1600s to 1700s held views that general health, mechanical injuries, trauma, and sudden temperature changes all caused caries—holding a common belief that decay was due to chemical agents, faulty saliva, and food particles. Until the early 1800s most writers believed that caries was due to inflammation from surrounding diseased alveolar bone. Todays science has demonstrated that caries is caused by indigenous oral microorganisms becoming a dynamic biofilm, that in the presence of fermentable sugars produce organic acids capable of dissolving inorganic enamel and dentin followed by the proteolytic destruction of collagen leaving soft infected dentin. As bacteria enter the pulp, infection follows.

Characteristics of Streptococcus mutans genotypes and dental caries in children

This longitudinal cohort study evaluated the diversity, commonality, and stability of Streptococcus mutans genotypes associated with dental caries history. Sixty-seven 5- and 6-yr-old children, considered as being at high caries risk, had plaque collected from baseline through 36 months for S. mutans isolation and genotyping using repetitive extragenic palindromic-PCR (4,392 total isolates). Decayed, missing, or filled surfaces (dmfs (primary teeth)/DMFS (secondary teeth)) for each child were recorded at baseline. At baseline, 18 distinct genotypes were found among 911 S. mutans isolates from 67 children (diversity), and 13 genotypes were shared by at least two children (commonality). The number of genotypes per individual was positively associated with the proportion of decayed surfaces (p-ds) at baseline. Twenty-four of the 39 children who were available at follow-up visits maintained a predominant genotype for the follow-up periods (stability) and this was negatively associated with the p-ds. The observed diversity, commonality, and stability of S. mutans genotypes represent a pattern of dental caries epidemiology in this high-caries-risk community, which suggests that fewer decayed surfaces are significantly associated with lower diversity and higher stability of S. mutans genotypes.

A randomized study of sodium hypochlorite versus formocresol pulpotomy in primary molar teeth.

BACKGROUND Alternatives to vital pulpotomy treatment in primary teeth are being sought because of the high formaldehyde content of traditional formocresol (FC) pulpotomy medicaments. AIM The aim was to compare the clinical and radiographic success of vital pulpotomy treatment in primary molars using 3% sodium hypochlorite (NaOCl) versus a 1:5 dilution of Buckleys FC. DESIGN Pulpotomies were performed in primary molars of healthy children between 3 and 10 years old. Sixty-five primary teeth were randomized into two groups that were evaluated for treatment outcomes. Following treatment, the pulp chamber was filled with zinc oxide eugenol (ZnOE) and restored with a stainless steel crown cemented with glass ionomer cement. Clinical and radiographic outcomes were recorded at 6 and 12 months. RESULTS The control (FC) and experimental (NaOCl) groups demonstrated 100% clinical success at 6 and 12 months. The NaOCl group had 86% (19/22) radiographic success at 6 months and 80% (12/15) at 12 months. The FC group had 84% (21/25) radiographic success at 6 months and 90% (9/10) at 12 months. No significant differences were found in the radiographic outcomes between the two groups at 6 and 12 months (Fishers exact test; P=0.574 and P=0.468, respectively). CONCLUSION NaOCl demonstrated clinical and radiographic success comparable to FC.

Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.

Comparison of preparation design and material thickness on microbial leakage through Cavit using a tooth model system

Few studies have compared Cavit thickness and access design as factors in microbial leakage. The present study used an acrylic tooth model to measure leakage of Streptococcus mutans. Pilot studies confirming the sterility of Cavit showed it will inhibit microbial growth for 2 days. The experiments compared class I preparations where Cavit thickness was 4 mm with class II preparations where thickness was 2-3 mm. Accesses sealed with cotton pellets were compared with those without cotton. Results of the study showed no bacterial contamination in any of the class I samples (up to 14 days). Some class II samples showed contamination at day 1 (3 out of 14), with all contaminated at day 7 (14 of 14), yet only 1 contaminated at day 14 (1 out of 14). The results suggest that a 4-mm thickness of Cavit should prevent bacterial ingress for at least 2 weeks, but microbial leakage may occur if temporary thickness is less than 3 mm or in a complex access preparation.