Detoxification of endotoxin by endodontic irrigants and calcium hydroxide.

The effects of endodontic irrigants and calcium hydroxide on lipopolysaccharide (LPS; endotoxin) were analyzed using the highly selective technique of mass spectrometry/gas chromatography with selected ion monitoring. An aqueous solution of LPS was mixed with one of a variety of endodontic irrigants for 30 min. Because it is a commonly used interappointment dressing, calcium hydroxide was also applied to LPS for 1, 2, or 5 days. LPS inactivation was measured by quantitation of free fatty acid release. Water, EDTA, ethanol, 0.12% chlorhexidine, chlorhexidine + sodium hypochlorite, and sodium hypochlorite alone showed little breakdown of LPS. Long-term calcium hydroxide--as well as 30-min exposure to an alkaline mixture of chlorhexidine, ethanol, and sodium hypochlorite--did detoxify LPS molecules by hydrolysis of ester bonds in the fatty acid chains of the lipid A moiety.

Effectiveness of three endodontic irrigants at various tubular depths in human dentin.

Bacteria from infected root canals can invade dentinal tubules, thus dentin disinfection is an important aspect of endodontic therapy. This study compares three endodontic irrigants for efficiency in killing bacteria established within human dentinal tubules. Root canals in extracted teeth were prepared and sterilized. Broth cultures of Enterococcus faecalis were allowed to grow within the canals to penetrate dentinal tubules. The infected canals were exposed individually to each of the irrigants for 1 min. Irrigants were 0.525% sodium hypochlorite, Tubulicid (0.2% EDTA), and 0.12% chlorhexidine (Peridex). Sterile water was the control. Viable bacteria were analyzed by drilling incrementally into dentin from the cementum toward the canal. Smaller diameter drills were used for each depth. Shavings were cultured at three depths, for each of three root levels: coronal, midroot, and apical. Although considerable variation occurred between roots, sodium hypochlorite seemed to be superior. Tubulicid and Peridex were better than water. More bacteria remained viable at greater distances from the pulp. These observations apparently apply to all levels in the canal.

Clearance of biofilms from dental unit waterlines through the use of hydroperoxide ion-phase transfer catalysts.

OBJECTIVES: The purpose of this research was to demonstrate the effectiveness of hydroperoxide ion-phase transfer catalyst (HPI-PTC) cleaners and disinfectants for maintaining dental unit waterlines free of planktonic organisms. METHOD AND MATERIALS: Water samples were taken from 117 sites, which included a variety of dental units and samples from the sink faucets of most operatories. Samples were plated on appropriate bacteriologic media and incubated. The presence or absence of biofilms was confirmed by scanning electron microscopy. Twenty-two of the dental units were retrofitted with independent water systems; the cleaning procedure involved an overnight application of an HPI-PTC cleaner followed by a 2-minute water rinse. RESULTS: Water from both the air-water syringe and the high-speed handpiece lines from all untreated units contained at least 6 x 10(2) colony-forming units per milliliter of planktonic or free-floating bacteria; the average was 1.4 x 10(5) CFU/mL. An initial 5% solution of HPI-PTC successfully cleared the lines of any apparent biofilm when applied for 3 consecutive days. Thereafter, once weekly use of the cleaner maintained the dental unit water supplies free of significant numbers of planktonic organisms. CONCLUSION: Routine weekly use of an HPI-PTC cleaner controlled dental unit waterline biofilm and reduced, with minimum effort, the microbial contamination level of water used for patient treatment to less than 200 CFU/mL.

Disinfection/sterilization of extracted teeth for dental student use.

Extracted human teeth are used in many preclinical courses. While there has been no report of disease transmission with extracted teeth, sterilization of teeth used in the teaching laboratory should be a concern. The purpose of this study was to determine the effectiveness of different sterilization/disinfection methods of extracted human teeth using Bacillus stearothermophilus, a bacteria resistant to heat and frequently used to test sterilizers. In this study, 110 extracted molars with no carious lesions were collected and stored in buffered saline. An endodontic occlusal access preparation was cut into the pulp chamber of each tooth. Pulp tissue in the chamber was removed with a broach. Approximately 1 x 10(5) B. stearothermophilus endospores in culture medium were injected into the pulp chamber, sealed with Cavit G, and then placed in sterile saline for twelve hours. Ten teeth were placed into each of eleven groups. Seven groups were immersed for one week in one of the following solutions: a) sterile saline (control group), b) 5.25% NaOCl, c) 2.6% NaOCl, d) 1% NaOCl, e) 10% buffered formalin, f) 2% gluteraldehyde, g) 0.28% quaternary ammonium. Four additional groups were treated by h) 10% formalin for two days, i) 10% formalin for four days, j) autoclaving at 240 degrees F and 20 psi for twenty minutes, and k) autoclaving at 240 degrees F and twenty psi for forty minutes. Each tooth was then aseptically split and placed in an individual test tube with growth medium. Samples were examined for evidence of growth (turbidity) at forty-eight hours. Only autoclaving for forty minutes at 240 degrees F and 20 psi or soaking in 10 percent formalin for one week were 100 percent effective in preventing growth. A chi-square analysis of the data indicates these two methods were significantly better than all other methods (p<0.001).

Special considerations for treating dental patients exhibiting the "post-polio syndrome".

A condition called "Post-polio Syndrome" (PPS) is a special type of neuromuscular disturbance that affects some elderly patients who had polio myelitis either as children or as young adults. It has been reported that approximately 1,600,000 polio survivors are alive today. Most will seek dental care, and up to half of the survivors will present with some form of PPS. This paper describes polio, its characteristics, and the long-term consideration of PPS, and discusses the special clinical implications related to this condition. Special emphasis is placed on physical impairments, breathing problems, and difficulty swallowing. Also included are sections discussing such topics as patient scheduling, office design and housekeeping, patient management, oral hygiene, diagnostic procedures, drug and pain management, and general health considerations.

In vitro disinfection of dentinal tubules by various endodontics irrigants.

Effectiveness of endodontic irrigants within dentinal tubules of human teeth was evaluated. Mid-sections of single-rooted teeth were prepared into dentin wedges. The pulpal sides of the sections were exposed to Micrococcus luteus or Bacillus megaterium that grew into the tubules. Irrigants used in the study included: 0.525% NaOCl, 0.12% chlorhexidine, RC Prep, 0.5% betadine iodine, and sterile H2O (as a control). Pulpal surfaces were exposed to an irrigant and then rinsed in sterile water. The samples were then cracked, exposing a fresh surface. Culture of the exposed dentin surfaces showed that selected irrigants reached to the far ends of the dentinal tubules in a concentration sufficient to kill 100% of the M. luteus. However B. megaterium was neither killed nor apparently inhibited by any irrigant. We conclude that endodontic irrigants permeate throughout dentinal tubules, but their effectiveness is dependent on the type of bacteria found within the tubules.

MRSA: an important consideration for geriatric dentistry practitioners.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause life-threatening disease in older populations. MRSA is readily spread by patient to patient or by health care workers to patient contact. Implications for dental practitioners include the fact that they may be passive vectors for the disease and that dental treatment of patients with active MRSA infections must follow effective infection control practices.

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity.

The admission index in the dental school admissions process.

Preprofessional students' grade point averages (GPAs) and aptitude test scores have been moderately successful in predicting student performance in dental school. The authors attempted to improve the predictability of the school's admission process by combining several preprofessional academic averages and selected nongraded personal attributes into a single Admission Index (AI) score. A Pearson r of 0.67 was found for the relationship between the AI and first-year dental school GPA for University of Louisville dental students accepted into the class of 1984. The correlation coefficient generated from the AI and first-year dental school GPA was markedly superior to those generated by any single predictor. The authors propose that the AI is of value not only for its use in the admission process, but also in the development of an interceptive student monitoring program for the less-qualified student.

Positive coooperativity in the binding of Streptococcus sanguis to hydroxylapatite.

The adherence of Streptococcus sanguis to hydroxylapatite beads has been analyzed by binding isotherms, Langmuir isotherms, and Scatchard plots. For saliva-coated beads, the Scatchard curves contained components with both positive and negative slopes. The results are interpreted as evidence for positive cooperativity in the binding process. Although all Scatchard curves were similar in shape, distinct differences were observed between saliva samples from different individuals. Salivary agglutinins against whole S. sanguis cells did not appear to influence the shapes of the curves or the extent of adherence. In addition, different strains of S. sanguis yielded similar Scatchard plots. When the binding of S. sanguis to buffer-coated hydroxylapatite beads was analyzed by Scatchard plots or binding isotherms, curves were generated which suggested that either direct ligand-ligand or nonspecific interactions were occurring. Hill plots of the adherence data yielded curves with slopes greater than unity for saliva-coated beads, providing additional support for the view that the interactions between S. sanguis and the pellicle involve cooperative phenomena. In contrast, a Hill plot for the binding data of S. sanguis to buffer-coated hydroxylapatite beads gave a curve with a slope of 0.91 +/- 0.07, suggesting negative cooperativity or limited specificity. When adherence data were plotted by the Langmuir method, curves were obtained which could not discriminate between the binding of the bacteria to the hydroxylapatite beads coated with either saliva or buffer. It was also observed that several different proteins and whole saliva tended to inhibit adherence. Scatchard plots, however, describing the binding of S. sanguis to the proteincoated beads were unique and revealed possible specific and nonspecific interactions. Scatchard analyses of binding data may be useful in understanding the mechanism(s) of adherence of streptococci to smooth surfaces.

Association of protein with the cell wall of Streptococcus mutans.

Cell walls from Streptococcus mutans were prepared by conventional technique and subjected to a series of extraction procedures involving classical protein solvents. The extracted walls contained several non-peptidoglycan amino acids and were also amenable to radiolabeling with [125I]sodium iodide and chloramine T. The cell walls could be chemically modified with tetranitromethane and diazo-1H-tetrazole, suggesting the presence of tyrosine or histidine or both. Flourescence spectra of the walls revealed the presence of either tyrosine or tryptophan. Several proteases, including pronase, trypsin, subtilisin, and proteinase K, removed some of the label from the walls. In contrast, treatment of the walls with salts or denaturants did not result in the solubilization of label. When the walls were solubilized with mutanolysin and subjected to chromatography, three peaks of radioactivity with apparent molecular weights of 73,000, 39,000, and 9,600 were observed. Wall digests subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band of radioactivity corresponding to an apparent molecular weight of 79,000. Isoelectric focusing of labeled wall digest gave rise to two major bands of radioactivity with isoelectric points of approximately 2.4 and 5.6. The results suggest that the cell wall of S. mutans contains tightly and possibley covalently bound polypeptide molecules. We propose that the cell wall polypeptides of S. mutans serve as factors in the attachment of the bacteria to smooth surfaces.

Streptococcus mutans adherence: presumptive evidence for protein-mediated attachment followed by glucan-dependent cellular accumulation.

Adherence of Streptococcus mutans to smooth surfaces has been attributed to the production of sucrose-derived d-glucans. However, several studies indicate that the bacterium will adhere in the absence of sucrose. The present data confirmed that S. mutans adherence to saliva-coated hydroxyapatite beads in the absence of sucrose is described by the Langmuir equation. The nature of the sucrose-independent adherence was studied with the Persea americana agglutinin as a selective adherence inhibitor. Pretreatment of the bacterium with P. americana agglutinin caused a 10-fold reduction in adherence, and the inhibition was not reversed with the addition of sucrose. Pretreatment of S. mutans with proteases also reduced adherence, regardless of the sucrose content, whereas periodate oxidation and glucanohydrolase treatment of the bacteria reduced sucrose-mediated adherence to the levels found for sucrose-independent adherence. The P. americana agglutinin, glucanohydrolase, and pepsin pretreatment of the cells did not eliminate sucrose-induced agglutination. Scanning electron microscopy showed that short streptococcal chains were bound to saliva-coated hydroxyapatite crystals in the sucrose-independent system, whereas the presence of sucrose caused larger bacterial clumps to be found. A two-reaction model of S. mutans adherence was developed from these data. It is proposed that one reaction is attachment to the tooth pellicle which is mediated by cell-surface proteins rather than glucans or teichoic acids. The other reaction is cellular accumulation mediated by sucrose-derived d-glucans and cell surface lectins. A series of sequential adherence experiments with P. americana agglutinin as a selective inhibitor provided presumptive evidence for the validity of our model of S. mutans adherence.

Lectin-like activity from Persea americana.

An extract from the seeds of Persea americana possessed an erythro-agglutinating activity. The agglutinin was devoid of specificity for carbohydrates, but interacted readily with basic proteins or basic polyamino acids. The interaction between the agglutinin and egg-white lysozyme was not inhibited by chaotropic salts, but was sensitive to relatively low concentrations of urea. An affinity chromatographic procedure was developed in an effort to purify the agglutinin. Products from the chromatographic procedure were found not to contain higher specific agglutinating activities than the crude extract. Amino acid acid analyses of the extract showed the presence of relatively high proportions of glutamic and aspartic acids. In addition, the extract contained phosphorus and a visible chromophore. The agglutinin was resistant to detergents and denaturants, and proteases, nucleases, and other enzymes. The results suggest that, as opposed to other plant agglutinins, the active component from Persea is not a protein. Similarly, in contrast to many lectins, the agglutinin from Persea was not mitogenic for mouse lymphocytes. The agglutinin partially inhibited the mitogenesis of lymphocytes when the cells were treated with concanavalin A, or with bacterial lipopolysaccharide.

Modification of in vitro adherence of Streptococcus mutans by plant lectins.

1) Several plant lectins inhibit in vitro adherence of S. mutans to smooth surfaces. 2) Initial attachment of S. mutans appears to be independent of sucrose-derived glucans. 3) Blockage of adherence is possibly due to lectin interaction with basic amino acid residues on the cell surfaces.

Analysis of the dextranase activity produced by an oral strain of Bacteroides ochraceus.

An anaerobic, gram-negative, dextranase-producing filamentous bacterium isolated from human dental plaque has been identified as a strain of Bacteroides ochraceus. The inducible intracellular dextran-degrading activities produced by this microoranism can be fractionated into endohydrolytic and exohydrolytic enzymes with distinct pH optima. These enzymes reduce the apparent rate of glucan production from sucrose by the dextransucrase produced by Streptococcus mutans and consequently may influence the in vivo production of polysaccharides involved in plaque accumulation and metabolism.

Inhibition of Streptococcus mutans strains by different mitis-salivarius agar preparations.

Several Streptococcus mutans strains were markedly inhibited by mitis-salivarius agar manufactured by Baltimore Biological Laboratories, but little, if any, inhibition was noted using Difco Laboratories' mitis-salivarius agar. Supplementation of the basic medium with sucrose and bacitracin for specific selection of S. mutans resulted in suppression of representative S. mutans type a strains regardless of manufacturer.

Characterization of a dextranase produced by an oral strain of Actinomyces israelii.

A dextranase-producing, gram-positive, anaerobic, rod-shaped bacterium isolated from human dental plaque was identified as Actinomyces israeli. Although the extracellular dextranase (EC 3.2.1.11) formed by this microbe appeared to be constitutively produced, the bacterium did not utilize the reaction products as a carbon source during growth. A striking feature of the dextranase was the formation of two distinct groups of oligosaccharide end products. The two groups presumably correspond to the limit dextran and the released reaction product which appeared to be cleaved from the end(s) of larger dextran molecules. Low levels of dextranase activity were measured by [3H]NaBH4 reduction and alcohol fixation of the large, tritiated end products on filter paper disks. Of the carbohydrate substrates tested, only alpha-1,6-linked glucans were cleaved. The enzyme did not exhibit any metal ion requirements, and its pH optimum was 6.3. It is suggested that the A. israelii dextranase may function as a regulatory factor during extracellular in vivo glucan synthesis from sucrose by various plaque microbes.

Dextranases from oral bacteria: inhibition of water-insoluble glucan production and adherence to smooth surfaces by Streptococcus mutans.

The effect of dextranases (EC 3.2.1.11) from the oral isolates Actinomyces israelii and Bacteroides ochraceus on water-insoluble glucan production by the Streptococcus mutans dextransucrase (EC 2.4.1.5) and sucrose-dependent adherence to smooth glass surfaces by S. mutans was studied. Collection on membrane filters of water-insoluble polysaccharides synthesized from radioactive sucrose was used to demonstrate the marked sensitivity of insoluble glucan formation to the presence of dextranase. Concentrations of A. israelii dextranase as low as 0.002 U/ml inhibited insoluble glucan formation by 60%. Similar results were obtained the the B. ochraceus enzyme. An assay for sucrose-stimulated adherence of S. mutans to smooth surfaces involved attachment of radioactively labeled nongrowing cells to the bottom of glass scintillation vials. This facile and sensitive assay was utilized to demonstrate that sucrose-dependent adherence was affected by low levels of dextranase from either A. israelii or B. ochraceus. Enzyme at 0.005 U/ml reduced adherence of S. mutans by 80%. Treatment of S. mutans cells previously attached to glass with low concentrations of the dextranases resulted in removal of 50% to 60% of the bacteria. The results indicate that dextranase-producing oral bacteria may affect sucrose-dependent colonization of S. mutans on the tooth surface and offer a possible explanation for both the difficulties involved in implanting this bacterium into the human mouth and the limited intraoral transmission of S. mutans from one tooth surface to another.