Roy H. Stevens

Evaluation of the antimicrobial potential of calcium hydroxide as an intracanal medicament.

Calcium hydroxide in the form of a supernatant liquid, as a slurry, or as Pulpdent was not as effective in destroying Streptococcus faecalis in the teeth of cats, or in vitro, as compared with camphorated chlorophenol.

Ability of bacterial endotoxin to diffuse through human dentin

An in vitro system was developed to determine whether bacterial endotoxin is capable of diffusing through dentin without the use of filtration pressure. Cavities were prepared in five third molar teeth in order to produce a split chamber device consisting of occlusal and pulpal chambers with 0.5 mm of intervening dentin. Endotoxin was introduced into the occlusal chamber and the effluent in the pulpal chamber was sampled every 30 min for 5 h and at 24 h using the limulus lysate assay. In four specimens the initial appearance of endotoxin in the effluent ranged from 15 min to 4 1/2 h. In two specimens the concentration of endotoxin in the effluent leveled off in 4 1/2 and 5 h, respectively, whereas in another two the concentration continued to increase throughout the experiment. In one specimen no endotoxin was detected. The results indicate that endotoxin is capable of passing through 0.5 mm of dentin.

Detection of cytotoxin genotypes of Helicobacter pylori in stomach, saliva and dental plaque

BACKGROUND The aim of this study was to detect the presence of Helicobacter pylori and its virulent cagA genes in the oral cavity of individuals with upper gastric diseases. Sixty-two individuals (42+/-2.3 years) with dispepsy symptoms, referred for gastroscopy and who were H. pylori positive in the gastric biopsy, were recruited and separated in two groups: case group-individuals with gastric disease (n = 30); control group-individuals with no gastric disease (n = 32); saliva, dental plaque and biopsy samples were collected from all individuals. Oral and biopsy samples were analyzed by PCR using specific primers for H. pylori 16S ribosomal and cagA genes. PCR products were sequenced for DNA homology confirmation. H. pylori was detected neither in dental plaque nor in saliva in the control group. In the case group H. pylori DNA was detected in 16/30 (53.3%) saliva samples and in 11/30 (36.6%) dental plaque samples. The cagA gene was detected in 13/30 (43.3%) gastric biopsies, in 7/16 (43.8%) saliva samples, and in 3/11 (27.3%) dental plaque samples. Eighteen (60.0%) individuals in the case group were H. pylori positive both in oral and biopsy samples, and 8 (26.6%) of those were positive for cagA-H. pylori DNA. H. pylori and its virulent clone showed a higher prevalence in the oral cavity of individuals in the case group than in the control group (p < 0.05). Our results suggest that dental plaque and saliva may serve as temporary reservoir for H. pylori and its virulent cagA variant in individuals with gastric disease.

Presence of Helicobacter pylori in supragingival dental plaque of individuals with periodontal disease and upper gastric diseases

BACKGROUND Helicobacter pylori is a Gram-negative microorganism which is able to colonize the gastric mucosa and is associated with peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Several studies have detected this bacterium in the oral cavity, suggesting it as a potential reservoir. The aim of this study was to investigate the presence of H. pylori in the oral cavity of individuals with periodontal disease and gastric diseases. METHODS 115 individuals, with mean age 49.6 (±5.8) years, were divided in 4 groups: (A) with gastric diseases and periodontal disease; (B) with gastric diseases and no periodontal disease; (C) without gastric diseases and without periodontal disease, (D) without gastric diseases and with periodontal disease. Supra and subgingival plaque samples were collected from posterior teeth of the individuals with sterile paper points, and prepared for Polymerase Chain Reaction analysis. Fishers exact test was used for detecting statistical differences between groups (p<0.05). RESULTS H. pylori was detected in supragingival plaque of 9/36 (25%) of group A, 1/31 (0.3%) of group B, 0 (0%) of group C and 3/36 (8.3%) of group D. No subgingival samples were positive for H. pylori. There was a statistically higher prevalence of H. pylori in groups A and D when compared to B and C (p<0.05). CONCLUSION H. pylori was detected in the supragingival plaque, but not in the subgingival plaque, of individuals with periodontal disease and upper gastric diseases. There was an association between the supragingival colonization of H. pylori and oral hygiene parameters such as the presence of plaque and gingival bleeding.

Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalis

INTRODUCTION Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections. METHODS Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis. RESULTS Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of NsiI and NdeI DNA fragments from one of the Siphoviridae phage isolates (phage phiEf11) indicated a genome size of approximately 41 kbp. CONCLUSION This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.

Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

The annotated complete DNA sequence of Enterococcus faecalis bacteriophage φEf11 and its comparison with all available phage and predicted prophage genomes

φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42,822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The genes were arranged in functional modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and possessed a predicted stem-loop structure in between predicted P(L) and P(R) promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers.

Higher levels of salivary MUC5B and MUC7 in individuals with gastric diseases who harbor Helicobacter pylori

OBJECTIVE The aim of the present study was to assess the salivary levels of MUC5B and MUC7 in individuals with dyspeptic disease and Helicobacter pylori (H. pylori) in the stomach, compared to individuals without dyspeptic disease. METHODS 30 individuals with dyspeptic disease, who underwent endoscopy for upper gastrointestinal complaints at Hospital Pedro Ernesto-RJ, Brasil and tested positive for H. pylori, and 23 controls with no dyspeptic disease, with mean age 53.5+/-4.4 years, were included in the study. Saliva samples and 3 antral biopsy were taken for PCR analysis and histologic examination. In addition, saliva samples were tested by ELISA with F2 monoclonal antibody and EU7A antibody against MUC7, to determine MUC5B and MUC7 levels, prior to endoscopic examination. The expression pattern of the proteins was quantified by comparison to a pooled saliva sample of 19 healthy volunteers. RESULTS MUC5B and MUC7 salivary levels were higher in the individuals with dyspeptic disease than in controls (p<0.0001). 33.3% (9/30) of the dyspeptic individuals and 0% of the controls had H. pylori in the oral cavity. CONCLUSIONS Individuals with gastric diseases, with H. pylori in the stomach, showed higher levels of salivary H. pylori receptors-MUC5B and MUC7-than individuals without gastric diseases. These results suggest that higher levels of specific salivary mucins could be useful as risk indicators for infection by H. pylori.

Genetic modifications to temperate Enterococcus faecalis phage Ef11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection.

Ef11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcus faecalis strain recovered from an infected root canal, and the Ef11 prophage is widely disseminated among strains of E. faecalis. Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by Ef11 as a first step in the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of Ef11 in E. faecalis JH2-2 yielded a variant that produced large, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between Ef11 and a defective FL1C-like prophage located in the E. faecalis JH2-2 chromosome. The recombinant possessed five ORFs of the defective FL1C-like prophage in place of six ORFs of the Ef11 genome. Deletion of the putative lysogeny gene module (ORFs 31-36) and replacement of the putative cro promoter from the recombinant phage genome with a nisin-inducible promoter resulted in no loss of virus infectivity. The genetic construct incorporating all the aforementioned Ef11 genomic modifications resulted in the generation of a variant that was incapable of lysogeny and insensitive to repressor, rendering it virulent and highly lytic, with a notably extended host range.

Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release

Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens.