A novel in vitro model system to grow films of oral bacteria for the study of human tooth root surface caries.

AIMS: To develop a simple and flexible novel in vitro model system to grow films of oral bacteria that could be used to study aspects of dental caries. METHODS AND RESULTS: Standardized suspensions of bacteria were inoculated into Ultrafree-CL (Millipore) ultrafiltration units at various densities. These were incubated for varying time intervals with a range of carbon sources. The bacterial films reproducibly achieved between 107 and 108 cfu cm-2, irrespective of the number inoculated and with no significant changes for 14 d. However, Streptococcus mutans grew through membranes with pores of diameter greater than 0.1 microm after 6 d. Culture of films in sucrose or water for 6 d led to a decreased number of colony-forming units, but returning them to broth reversed this. CONCLUSION: Reproducible films of oral bacteria can be cultured in Ultrafree-CL units. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that Ultrafree-CL units can be used as a simple model system to grow biofilms that could be used for dental caries research.

Scottish consortium for development and education in dental primary care.

A national consortium for dental primary care in Scotland has been formed as a result of integrated planning by groups involved in managing and delivering postgraduate dental education in Scotland. In 1998 a partnership was formed with representatives from the Scottish Council for Postgraduate Medical and Dental Education (SCPMDE) and the three Scottish dental institutions at Dundee, Edinburgh and Glasgow Universities. The principal aim of the Consortium is to promote evidence-based dental care through postgraduate education and research. The activities of the Consortium will provide a broadly based adjunct to current postgraduate provision with partner institutions contributing in different ways. Since its inception the Consortium has focused on two main areas: 'Higher Training' for Primary Care. Scottish Dental Practice Based Research Network.

Microbial factors and gingival crevicular fluid aspartate aminotransferase levels. A cross-sectional study.

The aim of this cross-sectional study was to investigate the clinical application of chairside tests for gingival crevicular fluid (GCF) aspartate aminotransferase (AST) levels and plaque BANA hydrolysis activity with the presence of the periodontal pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. The study comprised 100 periodontitis sites (pocket depths > or =4 mm, GI=3) from 10 patients with chronic adult periodontitis and 100 control sites (pocket depths <4 mm, GI<3) from 10 periodontally healthy patients comprising 55 healthy sites (pocket depths <4 mm, GI=0) and 45 gingivitis sites (pocket depths <4 mm, GI=1 or 2). The values for both BANA hydrolysis and AST levels were significantly higher in samples from periodontitis compared with gingivitis and healthy sites (p<0.001). A. actinomycetemcomitans was identified in 45% and P. gingivalis in 17% of periodontitis sites but neither pathogen was recovered from control sites and there was no significant correlation with the clinical parameters measured. There was no significant relationship between the presence of P. gingivalis and/or A. actinomycetemcomitans with BANA hydrolysis or AST levels. A significant correlation (p=0.0017) was observed between BANA hydrolysis and pocket depth and between AST hydrolysis and the GI (p=0.01). This study failed to demonstrate a positive association between chairside analysis of GCF metabolites for AST levels and/or BANA hydrolysis with P. gingivalis and A. actinomycetemcomitans. However, the GCF metabolites had a significant correlation with periodontally diseased sites in patients with chronic adult periodontitis and may help confirm clinical observations.

The in vitro adhesion of Candida albicans to buccal epithelial cells (BEC) from diabetic and non-diabetic individuals after in vivo and in vitro application of nystatin.

Buccal epithelial cells (BEC) from 12 patients with diabetes mellitus and 12 age- and sex-matched non-diabetic subjects were tested in vitro for adhesion of Candida albicans following exposure to nystatin both in vitro and in vivo. Adhesion was significantly reduced (P < 0.002) to cells from both the diabetic and non-diabetic subjects after in vitro exposure to nystatin, but the reduction in adhesion was variable (5.0-50.7% in control subjects and 0.5-48.4% in diabetic subjects) and equivalent between the two groups. In vivo exposure to nystatin produced no overall significant reduction in candidal adhesion to cells from either diabetic or control subjects.

Comparison of polymerase chain reaction and culture methods for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque samples.

In this study, the major periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were detected in subgingival plaque samples from patients with periodontal disease by polymerase chain reaction (PCR) and conventional culture methods. 170 plaque samples from 43 patients were analysed; A. actinomycetemcomitans and P. gingivalis were each detected in 40 (24%) of samples by PCR, whereas conventional culture methods detected A. actinomycetemcomitans and P. gingivalis in 25 (15%) and 18 (11%) of samples, respectively. The proportion of patients carrying A. actinomycetemcomitans in at least 1 sampled periodontal site was 17/43 (40%) by PCR and 13/43 (30%) by culture; for P. gingivalis this was 12/43 (28%) by PCR and 9/43 (21%) by culture. Only 5 samples, from 3 patients, harboured both A. actinomycetemcomitans and P. gingivalis. It is concluded that PCR is more accurate than conventional culture methods for identification of these periodontal pathogens in subgingival plaque samples and has a higher frequency of detection.

Altered taste sensation in newly-diagnosed NIDDM.

OBJECTIVE: To assess gustatory appreciation in newly diagnosed NIDDM patients and to determine whether it altered with the improvement of glycemic control after treatment with diet and oral hypoglycemic drugs. RESEARCH DESIGN AND METHODS: Assessments of taste, peripheral and autonomic neural function, diet, and oral microbiological flora were performed in 20 patients before and after treatment of hyperglycemia, 20 matched nondiabetic control subjects, and 11 patients with long duration of diabetes and advanced peripheral neuropathy. RESULTS: Median total HbA1 fell from 12.6 to 8.8% in new diabetic patients after 3-5 months of treatment. Electrical taste thresholds, detection threshold for glucose, and recognition threshold for glucose and salt were increased in newly-diagnosed NIDDM patients compared with the control subjects. The dose-response curve to glucose (using a visual analogue scale [VAS]) of newly-diagnosed NIDDM patients was significantly impaired and improved after treatment. By contrast, newly-diagnosed NIDDM patients had normal VAS taste responses to fructose, salt, and urea. Measurements of somatic and autonomic nerve function did not correlate with electrical or chemical taste function. CONCLUSIONS: Newly-diagnosed NIDDM patients have a blunted taste response, which displays a degree of specificity to glucose, is partially reversed after correction of hyperglycemia, and is independent of somatic or autonomic nerve function. This taste abnormality may influence the premorbid choice of nutrients, with a preference for sweet-tasting foods, thereby exacerbating hyperglycemia.

Anti-HIV antibody in saliva: an assessment of the role of the components of saliva, testing methodologies and collection systems.

The various components of saliva, namely mixed saliva, parotid saliva, submandibular saliva, crevicular fluid and minor (labial) gland secretions, were collected from 63 known HIV antibody seropositive patients. A commercial test system, Wellcozyme HIV 1+2, and an antibody capture ELISA (GACELISA), were compared for sensitivity against all components. Sensitivity of the GACELISA system was 100% in 123 mixed saliva, 121 parotid saliva and 127 labial fluid samples, and 98% in 99 submandibular samples and 127 crevicular fluid samples. Respective figures for Wellcozyme 1+2 were 92%, 55%, 73%, 66% and 63%. Mixed saliva was most easily, conveniently and effectively collected using a plain Salivette. In 241 Salivette samples examined from the 63 patients, GACELISA proved 100% sensitive, and Wellcozyme 95% sensitive. Another form of Salivette impregnated with citric acid was unsuitable for GACELISA and gave a false negative value of 45%. In 197 samples from the gingival margin taken by a dry swab, GACELISA showed a sensitivity of 98% and Wellcozyme 81%. The most sensitive method for demonstrating anti-HIV antibody in saliva is to collect mixed saliva with the plain Salivette system and assay anti-HIV antibody levels by GACELISA.

An evaluation of the Nd:YAG laser in periodontal pocket therapy.

The aim of this study was to determine whether the Nd:YAG laser energies of 50 and 80 mJ at 10 pulses per second (pps) were capable of improving the clinical parameters associated with periodontal disease. These energy settings were chosen as previous work indicated that higher values would damage root surfaces and that 80 mJ had an in-vitro bactericidal effect. Eighty periodontally affected sites in teeth scheduled for extraction from 11 patients with adult periodontitis were randomly placed in one of the following four treatment groups: 1. laser treatment at 50 mJ, 10 pps for 3 minutes; 2. laser treatment at 80 mJ, 10 pps for 3 minutes; 3. scaling and 4. untreated control. Probing depth, bleeding on probing (BOP), plaque index, gingival index and gingival crevicular fluid (GCF) volume were measured at baseline and week 6. Baseline subgingival microbiological samples were collected, then repeated immediately after treatment and at week 6 to assess the total anaerobic colony forming units (CFU). Only the scaling group showed a significant reduction in pocket depth and BOP (P < 0.001). The microbial samples taken immediately after scaling and laser at 80 mJ and 10 pps treatments showed a significant reduction in total CFU compared with the baseline (P < 0.01), which was sustained only in the scaling group until week 6. Electron microscopy did not reveal any heat damage on the root surfaces. This study demonstrated that application of Nd:YAG laser pulses of 50 mJ and 80 mJ failed to improve the clinical and microbiological parameters of periodontal disease.

Adherence of oral microorganisms to human parotid salivary proteins.

Bacterial colonisation of oral surfaces by microorganisms may be dependent on their interaction with specific host receptor molecules. Primary oral colonisers are known to remove specific proteins from parotid saliva. The aim of this study was to determine whether these interactions facilitate microbial attachment to a surface and hence identify specific salivary components as putative host receptor molecules. Parotid saliva was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. Suspensions of fluorescently labelled microorganisms were incubated with the blots and salivary components with adherent bacteria identified as fluorescent bands under ultraviolet (UV) transillumination. Species of streptococci known to be early colonisers of the clean tooth surface were found to adhere specifically to certain salivary proteins, especially to basic proline-rich proteins (PRPs). Polymorphic variations in these patterns could form the basis of differences in oral microflora, susceptibility to oral infections and consequent disease.

Oral disease in the elderly in long-term hospital care.

OBJECTIVE: To obtain baseline information on the prevalence of oral problems and disease in institutionalized elderly in a Scottish hospital. DESIGN: A cross-sectional clinical investigation with complementary microbiological studies as appropriate. SUBJECTS AND METHODS: A cohort of 147 elderly in five long-term care wards; collection of demographic data; clinical examination to determine: i) the dental and denture status and associated lesions such as Candida-associated denture stomatitis and angular cheilitis, ii) oral mucosal disease; swabs as appropriate for microbiology. MAIN OUTCOME MEASURES: Dental status, root caries prevalence; denture status and hygiene, and associated disease; oral mucosal health. RESULTS: Median period of institutionalization of 147 patients was 15 months and 65% were aged 80 years or more; the majority were significantly mentally impaired; 52% wore complete dentures, 9% were partially dentate, 19% possessed partial or incomplete dentures; 20% were neither dentate nor had dentures. Of 80 patients who verbalised their complaints, 35% complained of dry mouth and 45% had denture-related problems, principally discomfort. The commonest oral finding was a coated tongue (56%); angular cheilitis was present in 25%, and Candida-associated denture stomatitis in 19%; none had oral ulcers. CONCLUSIONS: There was considerable unmet dental need with significant oral disease and poor levels of oral and denture hygiene in this target group; both dental and medical professionals should act in concert to deliver curative as well as preventative dental care for the elderly living in institutions to improve their quality of life.

Microbiological assessment of denture hygiene among patients in longstay and daycare community places.

A simple, inexpensive microbiological assay for objective measurement of denture hygiene has been devised. Small individual filter paper discs were applied to the fitting surface of dentures for 20 seconds, eluted in phosphate buffered saline and bacterial counts on blood agar plates performed on the eluate. There was a strong positive correlation between denture cleanliness (graded on a scale of 0 to 4) and both total anaerobic count (r = 0.829; P < 0.001) and total aerobic count (r = 0.786; P < 0.001) following logarithmic transformation. Yeasts in the eluted disc specimens could be used as an aid to diagnose oral candidiasis. The method provided an objective measure of denture hygiene among elderly patients in longstay hospitals and daycare community places. It may be applicable to audit studies following implementation of new oral health care policies.

Evaluation of the in vitro cariogenic potential of Streptococcus mutans strains isolated from 12-year-old children with differing caries experience.

The limited usefulness of caries activity tests based solely on counts of mutans streptococci has been recognised increasingly. Such tests do not account for potential differences in cariogenicity of Streptococcus mutans strains harboured by individual patients. Hence, this study describes the evaluation of a simple and inexpensive method involving the dissolution of powdered hydroxyapatite as a means of determining the cariogenic potential of 64 freshly isolated strains of S. mutans from 24 children. Whilst it is apparent that differences between strains isolated from individuals could be demonstrated using this test, the variability observed was such that it was not possible to relate in vitro cariogenicity to actual clinical caries experience.

The in vitro proteolytic and saccharolytic activity of Candida species cultured in human saliva.

The proteolytic and saccharolytic activity of 4 Candida species was investigated in batch cultures of pooled, human mixed saliva supplemented with glucose. All the Candida species investigated (Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei) demonstrated a marked growth in saliva with a concomitant reduction in pH from about 7.5 to 3.3, within 72 h. Isotachophoretic analysis of the culture supernatant revealed the presence of a variety of acid anions of which pyruvate and acetate were the most abundant. Proteolysis of salivary components, evaluated by a biochemical assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was exhibited by all 4 Candida species, although there was inter-species variation. Despite the similarity in growth rates, C. tropicalis and C. krusei demonstrated greater proteolytic activity than C. albicans and C. glabrata. Neither candidal growth nor proteolysis was observed in glucose-free control saliva samples. In contrast, the degree of saccharolytic and proteolytic activity of a single isolate of C. albicans in glucose-supplemented parotid saliva appeared to be relatively weak compared with mixed saliva. As the oral cavity provides ideal low pH niches periodically supplemented with dietary carbohydrates, the acidic proteinases of Candida species may play a role in the pathogenesis of oral candidiasis.

The effect of indigenous bacterial populations on buccal epithelial cells on subsequent microbial adhesion in vitro.

Despite the numerous investigations on the adhesion of microorganisms to buccal epithelial cells, it is difficult to correlate different results, as disparate adherence values have been reported for a given organism. As one reason for this disparity may be the indigenous or natural bacterial populations on human buccal epithelial cells, the effect of the latter on subsequent microbial adherence in vitro was examined. There was a highly significant correlation between the degree of natural bacterial colonization on pooled buccal epithelial cells from 8 healthy donors and the adhesion of a single isolate each of Streptococcus mitis, Escherichia coli and Actinomyces naeslundii. However, no such relationship could be established for Candida albicans, Streptococcus milleri and another isolate of Streptococcus mitis. As in previous studies, variation in adherence values was found, both between samples from different donors, and from the same donor over time, but to a far lesser degree in pooled samples from different donors. These results imply that natural bacterial populations on buccal epithelial cells may affect the adhesion values derived from laboratory experimentation, and hence such data should be interpreted with caution.

The effect of nebulised pentamidine on the concentration of intra-oral Candida albicans in HIV-infected patients.

This prospective study investigated whether oral candidal carriage in HIV-infected patients was altered by pentamidine therapy. Repeated oral rinses were taken from 56 HIV-positive patients over a 2-year period. Oral candidal carriage was investigated in two groups of patients, one receiving prophylactic pentamidine therapy and the other not receiving regular prophylaxis. Patients receiving pentamidine had lower concentrations of Candida albicans intra-orally than patients who did not receive it. Furthermore, patients who received pentamidine at one stage of the study, but not another, also had lower concentrations of C. albicans intra-orally when receiving pentamidine. These findings indicate that pentamidine is useful as a local agent for prophylaxis against intra-oral candidosis in HIV-infected patients.

An in vitro investigation of the cariogenic potential of oral streptococci.

Whilst the importance of the mutans streptococci in the aetiology of dental caries is clear, a number of studies have described caries development in their absence. This investigation aimed to assess the cariogenic potential of Streptococcus gordonii, Strep. sanguis, Strep. vestibularis and Enterococcus faecalis in comparison with Strep. mutans and Strep. sobrinus, using a recently described in vitro model. In the presence of a 146 mM sucrose solution and powdered hydroxyapatite, each species was incubated for 5 h, after which acid anion production, calcium release and change in pH were measured. It was possible to assign each species to one of three groups, with E. faecalis, Strep. gordonii, Strep. sanguis exhibiting low, Strep. vestibularis intermediate, and Strep. mutans and Strep. sobrinus high in vitro cariogenic potential. This assay could be used as a screening test to assess the potential cariogenicity of a range of bacterial species.

Anti-Candida activity of murine bronchoalveolar lavage fluid.

Respiratory secretions provide an efficient method for protecting the large surface area of the lower respiratory tract. To determine whether lung secretions contribute to antifungal defences, murine bronchoalveolar lavage fluid (BLF) was tested for anti-candidal activity against 49 oral and vaginal isolates belonging to six different Candida species. The yeasts were incubated in unconcentrated, cell-free lavage fluid from Sprague-Dawley rats and then cultured quantitatively to measure residual viability. Experiments with C. albicans indicated that sensitivity to BLF increased in a time- and dose-dependent manner. This activity was heat-stable (56 degrees C) and consistent, irrespective of whether the BLF was derived from rats inoculated (orally) with candida or the uninoculated controls. Of the Candida spp. examined, C. albicans was the most susceptible followed by C. parapsilosis and C. tropicalis, whereas C. krusei, C. guilliermondii and C. glabrata were highly resistant. However, there were differences in susceptibility to BLF among different isolates within a given species. These results indicate that a heat-stable, soluble factor(s) in murine lavage fluid may suppress candidal colonisation of the lower respiratory tract and contribute to the defence mechanisms of the lungs.

The effect of exposure to chlorhexidine gluconate in vitro and in vivo on in vitro adhesion of Candida albicans to buccal epithelial cells from diabetic and non-diabetic subjects.

The effect of 0.2% chlorhexidine gluconate on in vitro adhesion of Candida albicans to buccal epithelial cells (BEC) was studied in 12 healthy subjects and 12 patients with diabetes mellitus. Exposure of BEC for one minute with 0.2% chlorhexidine gluconate in vitro, or by rinsing the mouth in vivo, resulted in a significant reduction in candidal adhesion to BEC in both diabetic and non-diabetic subjects and between both groups. In addition to the known fungicidal effect of chlorhexidine, it also reduces Candida albicans adhesion to oral mucosal cells, a factor of importance in the establishment of candidal infection.

Salivary protein interactions with oral bacteria: an electrophoretic study.

The relatively low levels of human salivary proteins in whole saliva as compared to the ductal secretions may be related to their interaction with oral bacteria. These interactions are thought to play an important role in the microbial colonisation of the mouth, and salivary proteins adsorbed to oral surfaces have been implicated in adherence. In this study we attempted to investigate the prevalence of interactive strains by screening a range of oral bacteria. Parotid saliva was incubated with bacterial suspensions and consequent alterations to the salivary protein profile determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A number of salivary components were found to be preferentially removed, particularly by those species known to be primary colonizers of clean tooth surfaces.

A randomised trial of co-amoxiclav (Augmentin) versus penicillin V in the treatment of acute dentoalveolar abscess.

In a randomised, operator-blind, comparative clinical trial, the efficacy of co-amoxiclav (250 mg amoxycillin plus 125 mg clavulanic acid, eight-hourly) was compared to that of penicillin V (250 mg phenoxymethylpenicillin, six-hourly) in the treatment of acute dentoalveolar abscess. In each case surgical drainage was established at the time of initial presentation by incision of any soft tissue swelling, accompanied by either extraction of the tooth or extirpation of the pulp as indicated clinically. Each patient recorded the severity of their pain and swelling at eight-hourly intervals using graduated scales. Clinical review was performed after 1, 2 and 5 days of treatment, at which time sublingual temperature was recorded and the presence of cervical lymphadenopathy determined. A total of 79 patients were clinically assessable on Day 5 (co-amoxiclav, n = 41; penicillin V, n = 38). The two groups were comparable for sex, age, presenting features and microbiology. Penicillin-resistant organisms were isolated from five patients (co-amoxiclav, n = 3; penicillin V, n = 2). Symptoms improved in all patients following the start of treatment, however those receiving co-amoxiclav recorded a significantly greater decrease in pain during the second (P = 0.026) and third days (P = 0.025). Only one patient reported a significant adverse effect associated with drug therapy, and this was in the penicillin group. Co-amoxiclav was as effective as penicillin therapy.