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.

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.

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.

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.

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.

Contribution of biofilm bacteria to the contamination of the dental unit water supply.

The authors studied the contribution of bacterial biofilm to the contamination of the dental unit water supply, as well as the effects of flushing and sodium hypochlorite treatment on reducing the number of contaminants. This study demonstrated that biofilm in the dental tubing was the primary source of contaminated water delivered by dental units.

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.

Bacterial contamination of the water supply in newly installed dental units.

Bacterial contamination of the water supply of newly installed dental units was investigated. Water samples were collected from water supply lines to the dental operatories prior to connection of the dental units. Within hours following connection, and continuing for up to the 6 months of the study, water samples were obtained from the air-water syringe of the units. The samples were serially diluted 10-fold and plated on culture media for quantitative analysis. The formation of bacterial biofilm in the dental water supply tubing was monitored by scanning electron microscopy. The results of these studies revealed that the building's water supply to the dental units was contaminated prior to connection to the units. The water supply from the air-water syringe was therefore contaminated as well. The number of contaminant bacteria in the dental unit water supply increased for several weeks and then stabilized. The lumen of the dental tubing became progressively contaminated with bacterial biofilm, which subsequently became the primary reservoir for maintenance of the contamination of the dental unit water supply.

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.

Bdellovibrios in Callinectus sapidus, the Blue Crab.

Bdellovibrios were recovered from the gill tissue of all of 31 crabs sampled and from all samples of epibiota obtained from the ventral shell surface of 15 crabs. The results suggest that the blue crab is a reservoir for bdellovibrios. The association with crabs may be an important factor in the ecology of the bdellovibrios.

Effects of temperature, salinity, and substrate on the colonization of surfaces in situ by aquatic bdellovibrios.

Recent studies suggest that surfaces are a more conducive habitat than the water column for the proliferation of bdellovibrios in the aquatic environment. The effect of temperature and salinity on the colonization of bdellovibrios on oyster shell, glass, and polystyrene surfaces in situ was investigated over an annual cycle. Sterile surfaces were suspended in various bodies of water for intervals ranging from 24 to 120 h. The results revealed that bdellovibrios associated with different types of surfaces over a broad temperature and salinity range. After 24 h of submersion in waters with temperatures from 9.0 to 26.7(deg)C, the ranges in log(inf10) values per square centimeter for the three surfaces were as follows: oyster shell, 2.2 to 2.5; glass, 0.3 to 2.2; and polystyrene, 0.7 to 1.6. Bdellovibrios were not recovered from surfaces submerged in water at temperatures below 8(deg)C during the 120-h experimental cycle. The number of bdellovibrios and culturable bacteria on oyster shells was significantly higher than the numbers on glass and polystyrene at all time intervals. The number of bdellovibrios was positively correlated with temperature and salinity on all surfaces. A positive correlation between the number of recoverable bacteria and temperature was observed, but the results with respect to salinity were diverse. The numbers of bdellovibrios recovered from oyster shells (up to 48 h) and water samples were significantly increased at salinities greater than 11(permil) compared to those in lower-salinity environments. The results of this study reveal that like many other bacteria in the aquatic environment, bdellovibrios prefer to associate with surfaces. This association provides the predators a rich source of prey bacteria in surface biofilms and perhaps protection in the gel-like matrix of the biofilm.

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients.

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.

Enhanced interleukin 1 beta, interleukin 6 and tumor necrosis factor alpha in gingival crevicular fluid from periodontal pockets of patients infected with human immunodeficiency virus 1.

Loss of periodontal support and eventually tooth loss is a common finding among acquired immunodeficiency syndrome (AIDS) patients. The cause of this destruction may be an increase in periodontal disease activity at sites within the same individual and also may be related to an increase in the pro-inflammatory cytokines, diffused through the gingival crevicular sulcus in AIDS patients. A study was undertaken to determine the relative levels of the pro-inflammatory cytokines, interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha), in gingival crevicular fluid collected from the deep (> 5 mm periodontal pocket depth) and shallow (< or = 3 mm periodontal pocket depth) periodontal pockets of 39 HIV-1-infected patients and 20 age-, race- and sex-matched uninfected controls. Complete medical history including risk factors such as intravenous drug abuse was taken. Gingival crevicular fluid samples were collected on periopaper strips. Cytokines were estimated by solid-phase enzyme-linked immunosorbent assay. To assess the degree of HIV activity, the viral load of these patients was determined by an Amplicor HIV-1 monitor kit using reverse transcriptase polymerase chain reaction. Gingival crevicular fluid from HIV-1-infected patients showed a two-fold increase in both IL-1 beta and TNF-alpha in deep periodontal pockets in comparison to shallow pockets, whereas IL-6 increased 1.8-fold. There was a significant (P < 0.05) increase in IL-1 beta, IL-6 and TNF-alpha in gingival crevicular fluid (both shallow and deep pockets) from HIV-1-infected patients in comparison to uninfected controls and also significantly elevated in deep versus shallow pockets in these patients. Although IL-1 beta, L-6 and TNF-alpha levels among HIV-1-infected patients with a high viral load (> 10,000 copies/ml) were higher than those from patients with a low viral load (< 400 copies/ml), only the increase in IL-1 beta level associated with deep pockets was significant (P < 0.05). There was also a trend of an increase in all the three cytokines among intravenous drug-abusing HIV-1-infected patients in comparison to non-intravenous drug abusers, but only the difference in IL-1 beta levels from deep pockets reached significance (P < 0.05). These enhanced pro-inflammatory cytokine levels in the gingival crevicular fluid of HIV-positive patients may be an important factor in causing the advanced periodontal lesions sometimes observed in HIV-positive patients.

Antigen activation of THP-1 human monocytic cells after stimulation with lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to evaluate the morphological changes in THP-1 cells and to measure expression of activation antigens (CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR) as evidence of maturation of THP-1 cells in response to stimulation by lipopolysaccharide (LPS) from the oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis, and granulocyte-macrophage colony-stimulating factor. THP-1 cells were stimulated with LPS (1 microgram/ml) of P. gingivalis or F. nucleatum for different time periods (1, 2, 4 and 7 d). Detection of different activation antigens on THP-1 cells was performed by indirect immunohistochemical staining followed by light microscopy. Confirmational studies were performed in parallel using indirect immunofluorescence and immunogold electron microscopy for detection of the corresponding activation antigens. Expression of different activation antigens by resting THP-1 cells revealed HLA-DR to be on 3% of the cells; CD-11b, 9%; CD-11c, 8%; CD-14, 22%; CD-35, 9% and CD-68, 7%. The CD-71 activation antigen was not expressed in untreated THP-1 cells. LPS stimulation increased expression of all activation antigens. A significant (p < 0.05) increase in expression of CD-11b, CD-11c, CD-14, CD-35, CD-68 and CD-71 was observed when GM-CSF (50 IU/ml) was supplemented during the treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis. Activation and differentiation of THP-1 cells by LPS from oral microorganisms in the presence of GM-CSF supports a role for human macrophages in acute and chronic periodontal diseases and may explain the clinically observable periodontal exacerbations in some patients after GM-CSF therapy.

Functional changes in THP-1 human monocytic cells after stimulation with lipopolysaccharide of oral microorganisms and granulocyte macrophage colony stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using F1TC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10-11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2-3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nucleatum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.

Retrospective identification and characterization of Candida dubliniensis isolates among Candida albicans clinical laboratory isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected individuals.

Fungal opportunistic infections, and in particular those caused by the various Candida species, have gained considerable significance as a cause of morbidity and, often, mortality. The newly described species Candida dubliniensis phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The present study was designed to determine the frequency at which this new species is not recognized in the clinical laboratory, to determine the patient populations with which C. dubliniensis is associated, to determine colonization versus infection frequency, and to assess fluconazole resistance. Over a 2-year period, 1,251 isolates that were initially identified as C. albicans by a hospital clinical laboratory were reevaluated for C. dubliniensis by inability to grow at 45 degrees C, colony color on CHROMagar Candida medium, coaggregation assay with Fusobacterium nucleatum, and sugar assimilation profiles (API 20C AUX yeast identification system). A total of 15 (1.2%) isolates from 12 patients were identified as C. dubliniensis. Ten of the patients were found to be immunocompromised (these included patients with human immunodeficiency virus infection or AIDS, cancer patients receiving chemotherapy, and patients awaiting transplantation). Thirteen isolates were highly susceptible to fluconazole (MIC, <0.5 microgram/ml). Three isolates from one patient, genotypically confirmed as the same strain, showed variable susceptibility to fluconazole. The first isolate was susceptible, whereas the other two isolates were dose-dependent susceptible (MIC, 16.0 microgram/ml). These data confirm the close association of C. dubliniensis with immunocompromised states and that increased fluconazole MICs may develop in vivo. This study emphasizes the importance of screening germ-tube-positive yeasts for the inability to grow at 45 degrees C followed by confirmatory tests in order to properly identify this species.

Coaggregation of Candida dubliniensis with Fusobacterium nucleatum.

The binding of microorganisms to each other and oral surfaces contributes to the progression of microbial infections in the oral cavity. Candida dubliniensis, a newly characterized species, has been identified in human immunodeficiency virus-seropositive patients and other immunocompromised individuals. C. dubliniensis phenotypically resembles Candida albicans in many respects yet can be identified and differentiated as a unique Candida species by phenotypic and genetic profiles. The purpose of this study was to determine oral coaggregation (CoAg) partners of C. dubliniensis and to compare these findings with CoAg of C. albicans under the same environmental conditions. Fifteen isolates of C. dubliniensis and 40 isolates of C. albicans were tested for their ability to coaggregate with strains of Fusobacterium nucleatum, Peptostreptococcus micros, Peptostreptococcus magnus, Peptostreptococcus anaerobius, Porphyromonas gingivalis, and Prevotella intermedia. When C. dubliniensis and C. albicans strains were grown at 37 degrees C on Sabouraud dextrose agar, only C. dubliniensis strains coaggregated with F. nucleatum ATCC 49256 and no C. albicans strains showed CoAg. However, when the C. dubliniensis and C. albicans strains were grown at 25 or 45 degrees C, both C. dubliniensis and C. albicans strains demonstrated CoAg with F. nucleatum. Heating the C. albicans strains (grown at 37 degrees C) at 85 degrees C for 30 min or treating them with dithiothreitol allowed the C. albicans strains grown at 37 degrees C to coaggregate with F. nucleatum. CoAg at all growth temperatures was inhibited by mannose and alpha-methyl mannoside but not by EDTA or arginine. The CoAg reaction between F. nucleatum and the Candida species involved a heat-labile component on F. nucleatum and a mannan-containing heat-stable receptor on the Candida species. The CoAg reactions between F. nucleatum and the Candida species may be important in the colonization of the yeast in the oral cavity, and the CoAg of C. dubliniensis by F. nucleatum when grown at 37 degrees C provides a rapid, specific, and inexpensive means to differentiate C. dubliniensis from C. albicans isolates in the clinical laboratory.

Identification of Candida dubliniensis in a prospective study of patients in the United States.

Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other yeast species are often identified in human immunodeficiency virus (HIV)-seropositive patients. Candida dubliniensis phenotypically resembles C. 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 the present study was to prospectively test for the presence of C. dubliniensis among clinical isolates and to determine the clinical and demographic characteristics of patients harboring C. dubliniensis. Over a 90-day period, isolates from 724 patients that were presumptively identified as C. albicans were screened for C. dubliniensis by use of tests for germ tube and chlamydospore production, by detection of an inability to grow at 45 degrees C, by colony color on CHROMagar Candida medium, and by the results of a sugar assimilation test with the API 20C AUX yeast identification system. Among 699 isolates retrieved from those specimens evaluated, 5 from 25 HIV-seropositive patients and 1 isolate from a patient whose HIV status was unknown were shown to be consistent by phenotyping and by electrophoretic karyotyping with the European reference strain of C. dubliniensis. One of the C. dubliniensis isolates had dose-dependent susceptibility to fluconazole (MIC, 16 microg/ml). These results confirm the presence of this interesting species in the United States and support the need for further investigations into the prevalence and pathogenesis of C. dubliniensis.

In vitro effect of oral antiseptics on human immunodeficiency virus-1 and herpes simplex virus type 1.

AIM: The antiviral effectiveness of widely used commercial mouthrinses has not been well studied. A project was undertaken to evaluate and compare the in vitro antiviral effectiveness of essential oil-containing mouthrinses (LA & TLA) and chlorhexidine mouthrinses (PX & CHX) on 2 different enveloped viruses, human immunodeficiency virus (HIV-1) and Herpes simplex virus (HSV-1) McIntyre strain. METHOD: HIV-1(89.6) (1x10(5)/ml) and HSV-1 (1x10(6)/ml) in RPMI-1640 medium were treated with two commercially available forms of LA & TLA (tartar control LA), and 2 formulations of chlorhexidine [(PX), 0.12% chlorhexidine & (CHX), 0.2% chlorhexidine] for 30 sec. The antiviral effect was estimated by inhibition of the syncytia formation or the cytopathic effect (CPE) for HIV-1 on MT-2 cells and by inhibition of the plaque formation for HSV-1 on Vero cell monolayers. RESULTS: Undiluted LA, TLA, PX and CHX completely inhibited both HIV-189.6 and HSV-1 McIntyre strain. PX and CHX inhibited HIV-1 up to 1:4 dilution, whereas, LA and TLA inhibited HSV-1 up to 1:2 dilution. The antiviral effects of LA and TLA were found to be similar and also the antiviral effect of PX and CHX were also found to be comparable. CONCLUSIONS: The methods used in this investigation allow easy and reproducible evaluations of antiviral efficacy. The anti-HIV-1 and anti-HSV-1 effects of LA, TLA, PX and CHX as evidenced in our in vitro study suggest that we should investigate potential in vivo effects during the use of essential oil-containing or chlorhexidine containing products when used by patients as mouthrinses. If the clinical studies confirm the in vitro data, pre-procedural use by clinicians may be beneficial in reducing viral contamination of bio-aerosols during the delivery of dental care.