The effect of a mouthrinse containing essential oils on dental restorative materials.

Mouthrinses that contain essential oils are effective for controlling plaque and periodontal disease. Recent studies have shown that such mouthrinses are effective at preventing the formation of biofilm in dental unit waterlines. However, there is no information in the literature regarding the effect of such mouthrinses on restorative materials used within the oral cavity. Specimens of three common restorative materials (a glass ionomer, a composite resin, and amalgam) were subjected to continuous exposure to Listerine and distilled water for 10 days; at that time, the strength, fluid sorption, and surface appearance of the specimens were compared. Specimens of the test materials also were placed in intraoral devices; volunteer patients wore these devices for 12 hours per day for a period of 10 days. During that time, the patients were instructed to rinse twice daily for 30 seconds with Listerine Cool Mint or a non-active mouthrinse. After 10 days, the specimens were salvaged from the devices and inspected by visible and SEM examination. This study indicates that routine use of mouthrinses containing essential oils (or even prolonged exposure to such mouthrinses) has no adverse effects on restorative materials that might be expected to react to such mixtures because of their chemical compositions. It was concluded that active mouthrinses do not appear to have any adverse effects on a variety of restorative biomaterials.

A system to maximize the in vitro immunological stability of human Langerhans cells.

Langerhans cells (LC) are now known to be immunocompetent antigen-presenting cells (APC) that are involved with numerous immune reactions initiated in skin, including the induction of contact hypersensitivity and the rejection of skin grafts. Immortalized cells that closely resemble in vivo LC have not been developed and mature LC, in vitro, appear to only briefly pass through a stage of differentiation when immunological potential seems stable. The literature supports the concept that mature LC possess numerous ultrastructural and antigen (Ag) characteristics. These include suface ATPase activity, Fc receptors, MHC class II Ag, CD1a Ag and Birbeck granules. These studies were undertaken to determine the ideal conditions of LC isolation and enrichment for persistence of these characteristics, such that toxicological and immunoreactive studies could closely mimic the in vivo situation. Studies were carried out to evaluate sources of tissue, duration of manipulations, chemical perturbation, stored v. fresh tissue, methods of enrichment, donor serological information and cost. LC were evaluated for ultrastructural and Ag stability by light and electron microscopy utilizing monoclonal antibodies (Mab) to surface Ag and ultrastructural features. It was determined that the most critical conditions included duration of manipulations and method of enrichment. Differences between fresh-frozen and fresh tissue samples were negligible. Cells evaluated less than 18 hr post-collection and enriched by density gradient centrifugation consistently demonstrated stability while ATPase activity, MHC Ag and ultrastructural characteristics varied when other methods were used. Implications for immunological and toxicological testing of in vitro skin samples can be drawn.

Cell surface hydrophobicity-associated adherence of Candida dubliniensis to human buccal epithelial cells.

Microbial adherence to mucosal surfaces is an important first step in the initiation of the pathogenic process in the oral cavity. Candida albicans, the most adherent and pathogenic Candida species, utilizes a variety of mechanisms to adhere to human tissues. Although the strongest mechanism of adherence involves mannoprotein adhesins on C. albicans, cell surface hydrophobicity (CSH) plays an important role in the adherence process by providing hydrophobic interactions that turn the initial attachment between the yeast and a surface into a strong bond. Recent cell wall analytical and comparative studies showed that, Candida dubliniensis, unlike C. albicans, possesses cell surface variations that allow it to be constantly hydrophobic, regardless of growth temperature. Based on these observations, the present study was designed to compare the adherence abilities of C. dubliniensis and C. albicans to pooled human buccal epithelial cells (BEC), in regards to their cell surface hydrophobicity. Ten C. albicans and nine C. dubliniensis isolates, as well as the C. albicans hydrophobic variant A9V10 were evaluated for adherence with BEC using visual aggregation in the wells of a microtiter plate and microscopic examination. All 11 C. albicans isolates failed to show adherence to BEC, visually or microscopically, when grown at 37 degrees C. The same isolates, however, showed significant increase in aggregation and microscopic adherence to BEC when grown at 25 degrees C. All C. dubliniensis isolates tested and the A9V10 C. albicans hydrophobic variant resulted in visual aggregation and adhered to BEC when grown at either temperature. The findings from this study show that, based on comparative adherence results and growth temperature changes, C. dubliniensis seems to have greater adherence to BEC than do typical C. albicans strains and that hydrophobic interactions seem to be the mechanism of adherence involved. Although many questions remain to be answered regarding the clinical implications of this observed in vitro enhanced adherence of C. dubliniensis to human BEC, these findings support the establishment of this novel species as a clinically significant yeast.

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation.

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.

In vitro studies of the efficacy of antimicrobials against fungi.

OBJECTIVE: The purpose of this study was to compare the efficacy of Listerine Antiseptic, Tartar Control Listerine Antiseptic, and Peridex mouthrinses and a 0.2% chlorhexidine digluconate solution against known pathogenic fungi. STUDY DESIGN: Standardized methods were used to compare the antimicrobial efficacy of the above agents versus representative fungal species. Minimum inhibitory concentration-minimum fungicidal concentrations in macrobroth dilutions, suspension kill-time, and effectiveness against an artificial biofilm-attached population were studied. RESULTS: All antimicrobials tested were effective against the fungal species under investigation at the concentration available commercially. Listerine Antiseptic showed a greater efficacy against attached artificial biofilm populations than the other antimicrobials tested. CONCLUSIONS: Listerine Antiseptic, Tartar Control Listerine Antiseptic, and Peridex mouthrinses show promise as a means to control the pathogenic fungal species under investigation and may have applications to reduce oral colonization.

Oral Candida dubliniensis as a clinically important species in HIV-seropositive patients in the United States.

OBJECTIVE: Interest in Candida dubliniensis has led to renewed clinical investigations regarding incidence, drug resistance, pathogenesis, and epidemiology of fungal infections in patients with HIV. C dubliniensis phenotypically resembles Candida albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of this study was to prospectively evaluate the prevalence of C dubliniensis in clinical isolates and determine the clinical and demographic characteristics of patients harboring C dubliniensis. STUDY DESIGN: Over a 6-week period, 24 yeast-positive isolates from HIV-positive dental patients were screened for C dubliniensis through use of phenotypic criteria. HIV viral load, CD4 count, and complete oral health evaluations were performed on each patient at the same visit during which the oral fungal surveillance culture was taken. RESULTS: Six isolates from 24 HIV-seropositive and yeast-positive patients were shown to be consistent phenotypically and by electrophoretic karyotyping with the European reference strain of C dubliniensis. Dose-dependent susceptibility to fluconazole was shown in one of the C dubliniensis isolates. Five of the 6 patients demonstrated moderate to high viral loads. General oral health, as evidenced by the presence of advanced periodontal lesions and a high decayed, missing, and filled teeth index (>20), was poor in 3 of the 6 patients with C dubliniensis and 7 of the 18 patients with C albicans. A history of intravenous drug abuse was present in 50% of the C dubliniensis -positive patients, which is representative of the HIV-positive population at the hospital. CONCLUSIONS: In this small sample, C dubliniensis represented 25% of the yeast-positive cultures. The clinical significance of this interesting species in the United States may be related to high viral load, rapid AIDS progression, and/or concomitant oral disease, such as a high caries index or periodontal disease.

Dental unit waterlines: biofilms, disinfection and recurrence.

BACKGROUND: Transmission of microbial pathogens to patients from biofilm within dental unit waterlines, or DUWLs, is a concern. To reduce the risk of toxicity to dental patients when water coolants are used, numerous chemical agents have been tested. In a series of trials, the authors investigated the recurrence of microbial growth after treating DUWLs with sodium hypochlorite (bleach), or B; glutaraldehyde, or G; or isopropanol 15.3 percent, or I. METHODS: The authors excised tubing sections from dental units in a general clinic. The tubing sections were evaluated at baseline and after overnight treatment. Effluent water samples and biofilm samples from tubing sections also were evaluated, by culture, at baseline and after treatment with the chemical agents. Biofilm within the tubing was examined by scanning electron microscopy, or SEM, and the authors identified bacterial isolates using standard techniques. The authors performed minimum inhibitory concentration tests on identified isolates pre- and posttreatment and compared the results to determine possible differences in resistance. RESULTS: In baseline evaluations, the authors determined that the effluent and biofilm matrix harbored an average of 1 x 10(5) colony-forming units, or CFU, per square centimeter and 1 x 10(4) CFU/cm2 recoverable microorganisms, respectively. A single overnight treatment of the DUWLs with B, G or I rendered effluent and biofilm samples that were free of recoverable bacteria. The number of viable bacteria in the effluent and the biofilm of B- or I-treated DUWLs returned to pretreatment levels by day six and day 15, respectively. DUWLs treated with G showed evidence of bacterial recurrence in the effluent and the biofilm to pretreatment levels by day three. The authors compared recurrence of biofilm and effluent posttreatment with untreated control tubing. The lower recurrence of viable bacteria in both biofilm and effluent samples for tubing treated with B and I was significant (P < or = .05). No evidence of resistance to the agents was noted during the study. Multiple treatments held the bacterial population to below recoverable levels but failed to remove the biofilm matrix, as evidenced by SEM. CONCLUSIONS: B, G and I eliminated recoverable bacteria after treatment and inhibited their recurrence in DUWL. Recolonization rates varied by agent. CLINICAL IMPLICATIONS: The residual effect of these agents raises concerns about the slow release of potentially toxic substances from the residual biofilm matrix. These agents reduce microorganisms in effluent water but do little to destroy the biofilm matrix in the DUWL, even with periodic treatments. Bacterial populations in the dental unit water rapidly recolonize the DUWL. Chemical agents or agents that potentially could be trapped in the matrix can represent an additional risk to the patient.

Effect of an antiseptic mouthrinse on salivary microbiota.

PURPOSE: To evaluate the effect of rinsing with original formulation Listerine Antiseptic (LA) on the level of viable salivary bacteria for periods up to 1 hour. MATERIALS AND METHODS: In this double-blind, controlled, cross-over study, unstimulated saliva was collected from 25 subjects, serially diluted, and cultured on selective and non-selective media under aerobic and anaerobic conditions. Streptococci, Veillonella sp., and total aerobic and anaerobic flora were enumerated just prior to and 2, 15, 30, and 60 minutes after rinsing for 30 seconds with either 20 ml of LA or a 5% hydroalcohol control rinse. RESULTS: After the control rinse, total flora cultivated on MM10 agar exhibited a non-significant (P > 0.05) 10%-20% decrease relative to baseline. In contrast, rinsing with LA resulted in a significant (P < 0.05) 60%-65% decrease from baseline in all four microbial groups at 2 minutes; except in the case of Veillonella, the significant decreases were sustained up to 60 minutes. Total Listerine group aerobic, anaerobic and streptococcal counts were significantly lower than placebo (P < 0.05). The significant reduction in salivary bacterial levels seen in the Listerine group for up to 60 minutes suggests that this antiseptic mouthrinse may have use clinically as a pre-procedural rinse to decrease the level of viable microorganisms in aerosols generated during dental procedures.

Identification of Candida dubliniensis in a study of HIV-seropositive pediatric dental patients.

PURPOSE: The combination of an immature immune system and suppressed cellular immunity in children with HIV infections provides optimal conditions for rapid disease progression. As a result, pediatric AIDS has become a major epidemiological challenge. Oral fungal colonization remains one of the most common opportunistic infections observed in both adult and pediatric HIV infected patients. Although Candida albicans is the most frequently isolated opportunistic fungal species, a recently characterized Candida species, C. dubliniensis, has gained considerable attention due to its almost exclusive association with HIV-seropositive individuals. The purpose of this study was to prospectively screen for the presence of C. dubliniensis among pediatric HIV+ patients. METHODS: Oral samples taken from twenty-seven children were cultured for the presence of yeast. All positive yeast isolates obtained were screened for the presence of C. dubliniensis by use of tests for germ tube and chlamydospore production, detection of inability to grow at 45 degrees C, by colony color on CHROMagar Candida medium, coaggregation with Fusobacterium nucleatum ATCC 49256 and by the results of sugar assimilation testing with the API 20C AUX yeast identification system. RESULTS: Among the 27 patients tested, 3 patients were found to harbor C. dubliniensis, one of which also grew C. glabrata; 12 patients were colonized with C. albicans, while the remaining 12 patients were negative for yeast. Identification of the three C. dubliniensis isolates was genetically confirmed by electrophoretic karyotyping. All three C. dubliniensis isolates were found to be susceptible to fluconazole (MIC < or = 0.25 ug/ml). CONCLUSIONS: These results confirm the presence of this novel species in a dental pediatric HIV seropositive population and support the need for further investigation into the prevalence and pathogenesis of C. dubliniensis.

Disinfection of dental unit waterlines with an oral antiseptic.

The problem of potential pathogens in biofilm within dental unit waterlines is real. Even though some chemical agents can disinfect biofilms, there remains concern that all remnants of the biofilm matrix are not eliminated, even with periodic treatments, and the bacterial populations in dental unit waterlines recur rapidly. Toxic and caustic residual chemicals are also a concern. In multiple trials following overnight treatment of dental unit waterlines with Listerine Antiseptic (LA), recurrence was investigated by evaluating effluent and biofilm specimens by plate culture. The presence or absence of biofilm within the dental unit waterlines was evaluated, pre- and post-treatment, by scanning electron microscopy. Baseline evaluations of dental unit waterlines determined the effluent and biofilm to harbor an average of 1 x 10(5) CFU per ml and 1 x 10(4) CFU per cm2, respectively, prior to treatment. Overnight, 18-hour treatment with LA rendered effluent and biofilm samples free of recoverable bacteria in all cases immediately following treatment. Viable bacteria in the effluent of treated dental unit waterlines recurred to near pre-treatment levels by Day 7. The minimum inhibitory concentrations for each of the recovered isolates did not change following overnight treatment. Repeated overnight treatments at the beginning of a one-week study were effective in inhibiting recurrence of viable bacteria in the biofilm and effluent indefinitely, but still failed to completely remove the biofilm matrix. New tubing treated prior to use and then daily with LA did not develop a detectable biofilm by scanning electron microscopy during the study. One-month long follow-up clinical trials have demonstrated that a maintenance solution of a 1:50 concentration of LA and sterile distilled water in self-contained dental units with new tubing is effective for prolonged periods in maintaining the effluent within the American Dental Association's recommendation for the year 2000 of < 200 CFU per ml. The clinical significance of these findings is that a solution to the problem of dental unit waterline contamination may be currently available. Since antimicrobial LA is safe for patient use, it may be one of the most viable options suggested to date.

Laboratory evaluation of anti-biofilm agents for use in dental unit waterlines.

Dental unit waterline biofilm has been recognized as a potential point of contamination and a risk to patients with any level of immunocompromise. Biofilm in dental unit waterlines, once established, has proven formidable to efforts in disinfection/disruption. This project compared standardized evaluation techniques by assessing the efficacy of a variety of agents that have been reported or suggested as useful in surface disinfection and/or antiseptic protocols. The zones of inhibition, minimum inhibitory/bactericidal concentrations and use-dilution with stainless steel carrier replicates tests assessed the disinfection of planktonic organisms using standardized microbial testing procedures. The disruption and/or disinfection of planktonic and biofilm organisms within naturally occurring dental unit waterlines were evaluated by culture and scanning electron microscopy. The six commercially available antimicrobial agents used to assess the techniques were bleach (sodium hypochlorite), Cavicide, glutaraldehyde, Listerine Antiseptic, Peridex and Sterilex Ultra. Comparisons between the results for each technique evaluated were determined for each product. All six agents demonstrated antimicrobial efficacy at the working concentrations designated by the manufacturers. Biofilm matrix elimination evaluated by scanning electron microscopy found virtually 0% elimination by glutaraldehyde to an estimated 90% elimination by Sterilex Ultra and bleach after one treatment. Treatment with Cavicide, Listerine Antiseptic and Peridex resulted in negligible elimination of the biofilm matrix. For comparability, the use of standardized testing techniques to evaluate a disinfection agent's efficacy against dental unit waterline contamination is essential. This project demonstrates a model system for evaluating disinfection agents potentially useful in the management of dental unit waterline biofilm, and should assist in educating the dental clinician in the appraisal of existing and future product claims.