The predominant Actinomyces spp. isolated from infected dentin of active root caries lesions.

Actinomyces are Gram-positive pleomorphic rods (GPPR) which form a large proportion of the oral microflora of all mammals. They have been implicated in root caries, although their role in dental caries initiation and progression is not well-understood. Many studies have focused on Actinomyces naeslundii, but few reports have documented other members of the GPPR. Therefore, we investigated the GPPRs isolated from infected dentin of active root caries lesions (n = 9) to determine which species were the most frequently isolated. The GPPR were isolated under both aerobic and anaerobic conditions and identified by biochemical and physiological tests to the species level according to the new taxonomy. Of 654 GPPR isolates investigated, 607 were identified as belonging to the genus Actinomyces. Of these, 242 were identified as A. israelii, 225 as A. gerencseriae, 109 as A. naeslundii, 15 as A. odontolyticus, and 13 as A. georgiae. Individual strains of A. israelii (n = 56) and A. gerencseriae (n = 46) were also investigated at the DNA level by means of Repetitive Extragenic Palindromic polymerase chain-reactions (REP-PCR) for the study of clonal diversity. Although only a small number of isolates was investigated, REP-PCR showed that the genotypes of both A. gerencseriae and A. israelii populations were heterogeneous within individual root caries lesions. A. gerencseriae and A. israelii strains from the same lesions did not share the same REP-PCR patterns, showing the robustness of the identification scheme. A significantly greater proportion of A. gerencseriae was isolated from the aerobic plates (p < 0.05), while the proportion of A. israelii was significantly (p < 0.05) greater from anaerobic plates. The role of individual Actinomyces spp. in the root caries process remains unclear, since various populations of GPPRs were isolated from individual active root caries lesions.

Dental caries, oral hygiene, and oral clearance in children with craniofacial disorders.

The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear. We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children. Children aged 6-16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied. Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group. The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001). The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group. However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm. The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

The predominant aciduric microflora of root-caries lesions.

The etiology of root caries is not fully understood, and although mutans streptococci, lactobacilli, and A. naeslundii have been implicated in its initiation and progression, this study was designed to determine the potential role of other microbial species and the nature of predominant aciduric microflora in the root caries process. We isolated the predominant aciduric microflora from root-caries lesions (n = 14) and sound root surfaces in subjects with (n = 13) or without (n = 10) root caries, using both a "most probable numbers" method and conventional plating methods. The predominant aciduric bacteria from root lesions were lactobacilli and A. israelii, while from sound root surfaces in subjects with root caries, A. gerencseriae comprised over 60% of aciduric isolates. Mutans streptococci were not among the aciduric isolates. Subjects without root caries harbored fewer bacteria, and S. anginosus (pH 4.8) and S. oralis (pH 5.2) were the predominant aciduric bacteria. The microbial etiology of root caries is more complex than was previously appreciated, and factors underlying the microbial succession occurring during the disease process are not known. Taxa with previously unrecognized aciduric characteristics have been isolated routinely, and the role of these organisms in the root caries process requires further investigation.

Dental caries and caries-associated micro-organisms in the saliva and plaque of 3- and 4-year-old Afro-Caribbean and Caucasian children in south London.

Three and four-year-old Caucasian and Afro-Caribbean children (n = 641) attending childcare facilities in the London boroughs of Lambeth, Lewisham and Southwark were studied in order to determine the prevalence of caries and caries-associated micro-organisms and the interactions between these for each of the racial groups; dmft (decayed, missing or filled teeth) scores were recorded using British Association for the Study of Community Dentistry criteria. Saliva and plaque samples were taken from each child and the numbers of mutans streptococci, lactobacilli and yeasts per sample were determined. The mean dmft scores of the 3- and 4-year-old Afro-Caribbean children were 0.36 +/- 0.10 and 0.51 +/- 0.13, respectively, compared to 0.80 +/- 0.17 and 1.48 +/- 0.24 for the equivalent Caucasian children (p < 0.001). Mutans streptococci and lactobacilli were recovered less frequently from the Afro-Caribbean children than from the Caucasian, but in both groups there were significant correlations between the plaque and salivary levels of mutants streptococci and caries experience. In both groups, children from whom both mutans streptococci and lactobacilli were isolated had the greatest mean dmft scores and these were not significantly different. Multiple regression analyses demonstrated that, after controlling for age and dmft, there were still significant associations between race and salivary levels of mutans streptococci and lactobacilli, p = 0.0013 and p = 0.0167, respectively. These data indicate that Afro-Caribbean children had lower levels of dental caries than Caucasian children living in the same London boroughs and attending the same preschool care facilities and, after controlling for age and caries experience, they also had lower salivary levels of mutans streptococci and lactobacilli.

The effects of the combination of chlorhexidine/thymol- and fluoride-containing varnishes on the severity of root caries lesions in frail institutionalised elderly people.

OBJECTIVES: To compare the clinical effects of a fluoride-containing varnish (Fluor-Protector) in combination with a chlorhexidine-containing varnish (Cervitec) on existing root caries lesions in a group of frail elderly subjects. METHODS: A randomised double blind longitudinal study was utilised. Subjects (n = 102) were randomly allocated to a Test or Placebo group. All leathery and soft root caries lesions in all subjects were coated with Fluor-Protector while the lesions in the Test group were also coated with Cervitec and the lesions in the Placebo group were coated with a Placebo varnish. Treatments were repeated five times in a 12-month period. Clinical parameters associated with root caries, measurements of individual lesions and salivary levels of caries associated bacteria were made at intervals. RESULTS: The clinical severity of the lesions in the Test group did not change significantly during the 12-month study period. In the Placebo group the mean lesion width and lesion height and length of exposed root increased significantly and the lesions were significantly closer to the gingival margin. There were no significant changes in the salivary levels of caries-associated microorganisms after 12 months although, in both groups, there was initially a significant reduction in the salivary levels of mutans streptococci. CONCLUSIONS: The combination of Fluor-Protector and Cervitec is a useful, simple, quick and non-invasive method for the control and management of existing root caries lesions. The procedure could be performed by a dental hygienist and may be usefully applied in other high-risk groups including persons with Parkinson's disease, debilitating neuromuscular conditions and dry mouth from whatever cause.

Does social deprivation in 1, 2, 3 and 4-year-old Scottish infants influence the frequency isolation of caries-associated micro-organisms?

OBJECTIVES: The aim of this study was to determine if there are associations between the level of social deprivation/affluence and the frequency isolation of caries-associated micro-organisms (Streptococcus mutans, Streptococcus sobrinus, lactobacilli and yeasts) in a large cohort of infants examined annually from 1 to 4 years of age. METHODS: DEPCAT was used to measure the socio-economic status of all consented infants (n=1099--1392) born in Dundee during a 1 year period (total n=1974). Caries-associated micro-organisms were cultured from saliva when the infants were 1, 2, 3 and 4 years of age. Standardised dental examinations were also carried out annually. Log linear analysis, which controlled for caries, was used to look for associations between DEPCAT and the isolation frequency of caries-associated micro-organisms. RESULTS: When controlling for caries, there was an association between DEPCAT and the isolation frequency of yeasts when the infants were 1 and 2 but not when 3 and 4 years old, whereas lactobacilli were associated only when the infants were 3 and 4 years old. Correlations between S. mutans and social deprivation were usually dependent on the caries status of the infants. CONCLUSIONS: The relationship between social deprivation and the isolation frequencies of caries-associated micro-organisms is complex with lactobacilli developing an association when the infants were 3 and 4 years old in contrast to yeasts which were only associated when the infants were 1 and 2 years old. Streptococcus mutans was associated with social deprivation when the infants were 2 years old and older, but dependent on caries status in the 3 and 4 year olds.

Effect of withdrawal of fluoride-containing toothpaste on the interproximal plaque microflora.

To compare the effects of fluoride-containing and fluoride-free toothpaste on plaque microflora, 15 subjects were enrolled in a double-blind crossover trial. All subjects used a fluoride toothpaste for 7 days before the trial started. Then, 4 interproximal sites per subject were professionally cleaned and subjects used one of the toothpastes for 5 days. On the 5th day plaque was collected from 2 sites, 12 and 6 h after toothpaste use. There was no difference between the groups in the numbers or proportions of aciduric bacteria (recovered at pH 4.8 or 5.2), or of yeasts, neisseriae, lactobacilli or streptococci (total or individual species, including Streptococcus mutans). However, the numbers and proportions of Gram-positive pleomorphic rods, primarily Actinomyces naeslundii, increased in 6-hour samples from subjects using fluoride toothpaste. The data suggest that the anti-caries effect of fluoride toothpaste is not mediated primarily through effects on the plaque microflora, although effects on plaque physiology could be important.

Phenotypic and genotypic selection of microbiota surviving under dental restorations.

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, beta-N-acetylglucosaminidase, and beta-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.

The microflora of the erupting first permanent molar.

The relationship between microflora, eruption status and caries status in the first permanent molar of young children was investigated in 177 children aged 6-7 years. A significantly greater proportion of fully erupted teeth were classified as sound and plaque-free compared to partially erupted teeth. Fully erupted teeth yielded greater numbers and proportions of mutans streptococci compared with significantly greater numbers and proportions of Actinomyces israelii in partially erupted teeth. Logistical regression analysis showed significant associations between white spot lesions in partially erupted teeth and increased numbers of Streptococcus oralis, mutans streptococci and Streptococcus salivarius whereas the presence of Actinomyces naeslundii was associated with health. Significantly greater numbers and proportions of S. oralis and S. salivarius were isolated from partially erupted teeth with white spot lesions whereas Streptococcus mutans was isolated in significantly greater numbers and proportions from fully erupted molars with white spots. This study suggests that organisms other than mutans streptococci are associated with caries development in erupting permanent molar teeth.

The isolation of Actinomyces naeslundii from sound root surfaces and root carious lesions.

The isolation of Actinomyces naeslundii from sound, exposed root surfaces (n = 56) and soft and leathery root carious lesions (n = 71) was investigated. Root carious lesions were sampled after the removal of overlying plaque. Supragingival plaque or carious dentine was sampled using a sterile excavator, the samples were disaggregated and cultured on both selective and non-selective media. A. naeslundii isolates were identified to the genospecies using specific antisera. Significantly greater numbers and proportions of A. naeslundii genospecies 2 than A. naeslundii genospecies 1 were isolated from all sites sampled. There was no significant difference between the numbers and proportions of the two genospecies isolated from leathery and soft lesions. The relationship between the presence of A. naeslundii genospecies and aciduric and acidogenic organisms was investigated. Those sound exposed root surfaces from which A. naeslundii genospecies 1 and/or 2 were isolated yielded significantly lower numbers of lactobacilli and yeasts than the surfaces from which A. naeslundii were not isolated. This difference was also found in leathery lesions but not soft root carious lesions. The microflora of soft root carious lesions was found to comprise primarily gram-positive pleomorphic rods which formed 70+/-7.8% of the flora, while in plaque from exposed root surfaces and in infected dentine from leathery lesions the gram-positive pleomorphic rods represented only 35% of the flora.

The influence of specific foods and oral hygiene on the microflora of fissures and smooth surfaces of molar teeth: A 5-day study.

A group of 20 students, harbouring >10(4) mutans streptococci per millilitre of saliva, was enrolled into the study. Models for sampling, reproducibly, the dental plaque present in specific sites (fissure and smooth surface) on the dentition were developed and validated. Withdrawal of normal oral hygiene procedures for only 1 day resulted in approximately 10-fold increases in the number of micro-organisms recovered from both sites. The effect of supplementing the subjects' diets with particular food items given 5 times per day [lemonade (5.8% w/v sugars, 250 ml), biscuits (digestive biscuits, 67.6% w/w carbohydrate of which 22% w/w was sugars and 45.6% w/w was starch), caramel toffees and sugar lumps] on the number of micro-organisms recovered and on the composition of the flora at both sites was determined. Dental plaque samples were taken after 5 days and it was found that supplementation of the diet with toffee and sugar lumps resulted in significantly more micro-organisms at both sampling sites. The supplementation of the diets with lemonade or biscuits did not significantly alter the numbers of micro-organisms recovered from either site. The percentage composition of the plaque samples from both dental sites remained relatively unaffected by oral hygiene although there were lower levels of mutans streptococci which might be related to the use of an antimicrobial toothpaste containing fluoride, triclosan and zinc citrate. This study suggests that the cariogenicity of certain sucrose-containing foods may, in part, be due to the enhancement of plaque accumulation in addition to other effects on the percentage composition of the plaque which may become manifest on prolonged usage of these dietary foodstuffs. These observations are consistent with dietary survey findings which often find consumption of confectionery related to caries experience or incidence.

The predominant microflora of nursing caries lesions.

The predominant microflora recovered from infected dentine of 52 carious teeth from 14 children with nursing caries was determined using both selective and non-selective media for the isolation of specific genera and acidified media (pH 5.2) to isolate the predominant aciduric microorganisms, and compared with the microflora of sound enamel surfaces in caries-free children. Streptococcus mutans formed a significantly greater proportion of the lesion flora while Streptococcus oralis, Streptococcus sanguis and Streptococcus gordonii formed a significantly greater proportion of the plaque flora from sound tooth surfaces. The proportions of Actinomyces naeslundii and Actinomyces odontolyticus were significantly greater in the plaque samples than in the lesion samples. Actinomyces israelii formed 18.2% of the flora from the lesions, but was not isolated from the plaque samples. The proportions of Candida albicans, Lactobacillus spp. and Veillonella spp. were also significantly greater in the carious dentine than in the plaque samples. The most frequently isolated lactobacilli were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus rhamnosus. The predominant aciduric flora was S. oralis, S. mutans and A. israelii and these taxa were also isolated from a similar proportion of the lesions at pH 7.0. Strains of S. mutans, L. casei, L. fermentum and L. rhamnosus isolated from individual carious teeth were genotyped using PCR-based methods. Each species was genotypically heterogeneous and different genotypes were recovered from different carious teeth in the same child. These data indicate that the microflora of lesions in the same child is microbiologically diverse and support a non-specific aetiology for nursing caries in which the physiological characteristics of the infecting flora, not its composition, is the major determinant underlying the disease process.

Comparison between visual examination and a laser fluorescence system for in vivo diagnosis of occlusal caries.

This study compared a laser fluorescence (LF) system (DIAGNOdent) with a visual caries scoring system for in vivo detection and diagnosis of occlusal caries under the conditions of an epidemiological study, in 132 mandibular and 38 maxillary first permanent molars in 170 children (mean age: 6.85 +/- 0.58 years). The teeth were cleaned and occlusal caries status in a selected investigation site recorded using both visual and LF systems. The LF readings were interpreted both according to the cut-off points recommended by the manufacturers and those based on laboratory research with histological validation. The percentage agreement of the LF and visual scoring system was better using the cut-off limits recommended by the manufacturer. Histological validation was not possible in this clinical study, but it appeared that either the LF method was overscoring some lesions or the visual method was underscoring them. Since the LF instrument cannot be expected to differentiate caries from hypomineralisation, it should probably be used as an adjunct to a clinical examination.

Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque.

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (chi(2) = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.

Use of CHROMagar Candida medium for isolation of yeasts from dental samples.

A new differential medium, CHROMagar Candida, for the isolation of clinically important yeasts was investigated to determine its usefulness in facilitating the study of oral yeasts. The recovery of yeasts on the medium was not significantly different from the recovery on Sabouraud dextrose agar. The identities of 450 green colonies on CHROMagar Candida, presumptively identified as Candida albicans on the basis of the manufacturer's instructions, were confirmed by testing for beta-N-acetylgalactosaminidase. Candida tropicalis also formed distinctive colonies, and other yeasts including Candida (Torulopsis) glabrata, Candida Parapsilosis, Candida Magnoliae, Candida lusitaniae, Candida Famata, Candida kefir, and Saccharomyces cerevisiae were readily distinguished from C. albicans and C. tropicalis isolates. CHROMagar Candida is a very useful medium, and its use will facilitate the study of yeasts associated with dental diseases.

Intra-oral acid production associated with eating whole or pulped raw fruits.

The hypotheses that raw fruits, whether whole or pulped, were cleared rapidly from the mouth and that the sugars in the whole and pulped fruits are fermented with equal efficiency to acids by the oral microflora were tested in this study. Groups of 7-9 adult subjects chewed 10 g of raw, whole or pulped fruit (apple, banana, orange, pear and pineapple) for 1 min and whole, unstimulated saliva samples were collected during the following 60-min interval. Each saliva sample was assayed for the concentrations of fruit-derived sugars (glucose, fructose and sucrose), fruit-derived acids (malic and citric) and acids which may be produced as a result of bacterial fermentation (acetic, lactic, formic and succinic). We found the fruit-derived sugars were rapidly cleared from the mouth (within 5 min). The major bacterially produced acids were lactic and succinic, which reached maximum concentrations in the 5-min sample. There was no significant difference, within a fruit, in the salivary levels of any of the sugars or acids between the raw whole or raw pulped forms. In light of these findings it seems unwise to assume that fruits may be consumed without consideration of their acidogenic potential.