David Beighton

A scheme for the identification of viridans streptococci.

A collection of strains representing all the currently recognised species of oral or viridans streptococci (Streptococcus sanguis, S. parasanguis, S. gordonii, S. oralis, S. mitis, S. salivarius, S. vestibularis, S. anginosus, S. constellatus, s. intermedius, S. mutans and S. sobrinus) were tested for the production of a range of glycosidase activities with 4-methylumbelliferyl-linked fluorogenic substrates, and for reactions in a range of conventional fermentation and hydrolytic tests. The resulting biochemical scheme, consisting of 14 tests, enabled the differentiation of all these species and distinguished three biotypes within S. sanguis. The scheme reported here represents an improvement over currently available schemes for the identification of viridans streptococci.

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.

The Bifidobacterium dentium Bd1 Genome Sequence Reflects Its Genetic Adaptation to the Human Oral Cavity

Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.

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.

Marginal Ditching and Staining as a Predictor of Secondary Caries Around Amalgam Restorations: A Clinical and Microbiological Study

Caries at the margins of restorations is difficult to diagnose. In particular, the relevance of both marginal ditching and staining around amalgam restorations is unclear. This clinical study questions the relevance of marginal ditching and color change to the level of infection of the dentin beneath the margins of amalgam restorations. Clinically visible sites (330) on the tooth/restoration margin were selected on 175 teeth. The enamel adjacent to each site was noted as stained (a grey discoloration) or stain-free. One hundred and seventy-eight sites were clinically intact, 83 sites had narrow ditches (< 0.4 mm), and at 49 sites, wide ditches were present (> 0.4 mm). Twenty sites with frankly carious lesions were also included. Plaque was sampled at the tooth-restoration margin, and the dentin was sampled at the enamel-dentin junction below each site. Samples were vortexed, diluted, and cultured for total anaerobic counts, mutans streptococci, lactobacilli, and yeasts. Plaque samples showed that margins with wide ditches (> 0.4 mm) harbored significantly more bacteria, mutans streptococci, and lactobacilli than did clinically intact margins and margins with narrow ditches. There were no significant differences in the degree of infection of the dentin beneath clinically intact restorations and those with narrow ditches, but samples associated with wide ditches and carious lesions yielded significantly more bacteria, mutans streptococci, and lactobacilli. The color of the enamel adjacent to the sample site was irrelevant to the level of infection of the dentin beneath the filling margin, provided a frankly carious lesion was not present. The results suggest that amalgam fillings where margins show wide ditches or carious lesions should be replaced. Narrow ditches and color change alone should not trigger the replacement of a filling.

Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions

ABSTRACT Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.

Prediction of Secondary Caries around Tooth-colored Restorations: A Clinical and Microbiological Study

Caries at the margins of restorations is difficult to diagnose, and the relevance of staining and ditching around tooth-colored fillings is unclear. This clinical study questions the relevance of marginal color change and marginal ditching to the level of infection of the dentin beneath the margins of tooth-colored restorations. Clinically visible sites (197) on the tooth/restoration margin were selected in 113 teeth. The filling margin and the enamel adjacent to each site were noted as stained or stain-free, and sites were graded as intact, having a narrow ditch, or having a wide ditch. Thirty sites with frankly carious lesions were also included. Plaque was sampled at the toothrestoration margin and the filling removed. The enamel-dentin junction (EDJ) at each sample site was noted as hard or soft when probed, and the dentin was sampled. Samples were vortexed, diluted, and cultured for total anaerobic counts, mutans streptococci, and lactobacilli. There were more bacteria in the plaque over frankly carious cavities, and the dentin was soft and heavily infected. Only 38 out of 167 sites without frankly carious cavities had soft dentin at the EDJ. Both the plaque and dentin in these sites harbored more micro-organisms. However, none of the clinical criteria chosen would reliably predict the presence of this soft dentin. In this study, only a frankly carious lesion at the margin of the filling constituted a reliable diagnosis of secondary caries.

Mechanisms of Resistance to Quinupristin-Dalfopristin among Isolates of Enterococcus faecium from Animals, Raw Meat, and Hospital Patients in Western Europe

ABSTRACT Twenty-eight quinupristin-dalfopristin-resistant isolates ofEnterococcus faecium from hospital patients and nonhuman sources in European countries were studied. High-level resistance (MICs, ≥32 μg/ml) was associated with the presence ofvat(E) (satG) (14 isolates [50%]) orvat(D) (satA) (6 isolates [21%]). These genes were not detected in eight (29%) isolates with lower levels of quinupristin-dalfopristin resistance (MICs, 4 to 16 μg/ml). This suggests the presence of further mechanisms of resistance to quinupristin-dalfopristin in E. faecium.

Oral health of elderly occupants in residential homes

1We offered to assess the oral health of elderly occupants in a random sample of 55 of the 110 residential and nursing homes in West Hertfordshire. 48 homes accepted. We recorded participants’ age, sex, length of time in the home, medications, number of medicines administered as syrups, and type of dentures worn. Extraoral and intraoral soft tissues, denture hygiene status, clinical status of each coronal and root surface, plaque and gingival indices were examined and recorded. A questionnaire that covered oral health and past dental care was completed in the form of a structured interview and was conducted with all those residents who were able to respond. The sample consisted of 249 men and 792 women, with a mean age of 83·9 (SD 7·8) years, who had spent 24·9 (26·2) months in the residential homes. 598 residents were edentulous, 203 residents had only teeth, and 240 had teeth and dentures. The results (table) are consistent with findings in other institutionalised populations.

Dental Caries from a Molecular Microbiological Perspective

Dental caries results from an imbalance of the metabolic activity in the dental biofilm. The microbial communities of teeth have traditionally been studied by standard cultural approaches. More recently, cloning and sequencing of the 16S rRNA gene have been used to characterize the microbial composition of the oral biofilm, but the methodological limitations of this approach have now been recognized. Next-generation high-throughput sequencing methods have the potential to reveal the composition and functioning of the biofilm by means of metagenomic and metatranscriptomic analyses. Currently available high-throughput sequencing approaches are reviewed and discussed in relation to studying the biofilm associated with dental caries. Important in understanding the dynamic processes in caries is the metabolic activity of the biofilm; metabolome analysis is a new tool that might enable us to assess such activity. As caries is a localized disease, it is essential that biofilm samples are taken from precisely determined tooth sites; pooling samples is not appropriate. This paper presents the case that culture-based studies are important, but that the fullest understanding of the role of the biofilm in the caries process will only come from an integrated approach determining biological function and metabolic output.