Conservative Management of Dental Caries Using Silver Nitrate and Atraumatic Restorative Treatment in Remote Rural Setting.

Managing dental caries remains a challenge to clinicians­more so when treating caries in remote, underserved areas of the world. The purpose of this article is to report preliminary results of an effort to reduce to practice the best features of silver nitrate (SN) anti-caries effect and atraumatic restorative treatment (ART) for lesion preparation and restoration. The modifications made to two approaches have practical applications to school-based outreach programs hoping to respond to the dire need for preventive and conservative restorative methods in parts of the world where access to the dental office is unobtainable.

Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium.

Like Streptococcus mutans, lactobacilli are commonly isolated from carious sites, although their exact role in caries development remains unclear. This study used mixed-species models to analyze biofilm formation by major groups of oral lactobacilli, including L. casei, L. fermentum, L. rhamnosus, L. salivarius ssp. salivarius, and L. gasseri. The results showed that lactobacilli did not form good biofilms when grown alone, although differences existed between different species. When grown together with S. mutans, biofilm formation by L. gasseri and L. rhamnosus was increased by 2-log (P < 0.001), while biofilms by L. fermentum reduced by >1-log (P < 0.001). L. casei enhanced biofilm formation by ~2-log when grown with S. mutans wild-type, but no such effects were observed with S. mutans deficient of glucosyltransferase GtfB and adhesin P1. Both S. mutans and L. casei in dual-species enhanced resistance to acid killing with increases of survival rate by >1-log (P < 0.001), but drastically reduced the survival rates following exposure to hydrogen peroxide (P < 0.001), as compared to the respective mono-species cultures. When analyzed by RNA-seq, more than 134 genes were identified in S. mutans in dual-species with L. casei as either up- or down-regulated when compared to those grown alone. The up-regulated genes include those for superoxide dismutase, NADH oxidase, and members of the mutanobactin biosynthesis cluster. Among the down-regulated genes were those for GtfB and alternative sigma factor SigX. These results further suggest that interactions between S. mutans and oral lactobacilli are species-specific and may have significant impact on cariogenic potential of the community.

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.

Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children's medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.

Comparative genomics of oral isolates of Streptococcus mutans by in silico genome subtraction does not reveal accessory DNA associated with severe early childhood caries.

Comparative genomics is a popular method for the identification of microbial virulence determinants, especially since the sequencing of a large number of whole bacterial genomes from pathogenic and non-pathogenic strains has become relatively inexpensive. The bioinformatics pipelines for comparative genomics usually include gene prediction and annotation and can require significant computer power. To circumvent this, we developed a rapid method for genome-scale in silico subtractive hybridization, based on blastn and independent of feature identification and annotation. Whole genome comparisons by in silico genome subtraction were performed to identify genetic loci specific to Streptococcus mutans strains associated with severe early childhood caries (S-ECC), compared to strains isolated from caries-free (CF) children. The genome similarity of the 20 S. mutans strains included in this study, calculated by Simrank k-mer sharing, ranged from 79.5% to 90.9%, confirming this is a genetically heterogeneous group of strains. We identified strain-specific genetic elements in 19 strains, with sizes ranging from 200 to 39 kb. These elements contained protein-coding regions with functions mostly associated with mobile DNA. We did not, however, identify any genetic loci consistently associated with dental caries, i.e., shared by all the S-ECC strains and absent in the CF strains. Conversely, we did not identify any genetic loci specific with the healthy group. Comparison of previously published genomes from pathogenic and carriage strains of Neisseria meningitidis with our in silico genome subtraction yielded the same set of genes specific to the pathogenic strains, thus validating our method. Our results suggest that S. mutans strains derived from caries active or caries free dentitions cannot be differentiated based on the presence or absence of specific genetic elements. Our in silico genome subtraction method is available as the Microbial Genome Comparison (MGC) tool, with a user-friendly JAVA graphical interface.

Phylogenetic analysis of glucosyltransferases and implications for the coevolution of mutans streptococci with their mammalian hosts.

Glucosyltransferases (Gtfs) catalyze the synthesis of glucans from sucrose and are produced by several species of lactic-acid bacteria. The oral bacterium Streptococcus mutans produces large amounts of glucans through the action of three Gtfs. GtfD produces water-soluble glucan (WSG), GtfB synthesizes water-insoluble glucans (WIG) and GtfC produces mainly WIG but also WSG. These enzymes, especially those synthesizing WIG, are of particular interest because of their role in the formation of dental plaque, an environment where S. mutans can thrive and produce lactic acid, promoting the formation of dental caries. We sequenced the gtfB, gtfC and gtfD genes from several mutans streptococcal strains isolated from the oral cavity of humans and searched for their homologues in strains isolated from chimpanzees and macaque monkeys. The sequence data were analyzed in conjunction with the available Gtf sequences from other bacteria in the genera Streptococcus, Lactobacillus and Leuconostoc to gain insights into the evolutionary history of this family of enzymes, with a particular emphasis on S. mutans Gtfs. Our analyses indicate that streptococcal Gtfs arose from a common ancestral progenitor gene, and that they expanded to form two clades according to the type of glucan they synthesize. We also show that the clade of streptococcal Gtfs synthesizing WIG appeared shortly after the divergence of viviparous, dentate mammals, which potentially contributed to the formation of dental plaque and the establishment of several streptococci in the oral cavity. The two S. mutans Gtfs capable of WIG synthesis, GtfB and GtfC, are likely the product of a gene duplication event. We dated this event to coincide with the divergence of the genomes of ancestral early primates. Thus, the acquisition and diversification of S. mutans Gtfs predates modern humans and is unrelated to the increase in dietary sucrose consumption.

Distribution of putative virulence genes in Streptococcus mutans strains does not correlate with caries experience.

Streptococcus mutans, a member of the human oral flora, is a widely recognized etiological agent of dental caries. The cariogenic potential of S. mutans is related to its ability to metabolize a wide variety of sugars, form a robust biofilm, produce copious amounts of lactic acid, and thrive in the acid environment that it generates. The remarkable genetic variability present within the species is reflected at the phenotypic level, notably in the differences in the cariogenic potential between strains. However, the genetic basis of these differences is yet to be elucidated. In this study, we surveyed by PCR and DNA hybridization the distribution of putative virulence genes, genomic islands, and insertion sequences across a collection of 33 strains isolated from either children with severe early childhood caries (S-ECC) or those who were caries free (CF). We found this genetically diverse group of isolates to be remarkably homogeneous with regard to the distribution of the putative virulence genes and genetic elements analyzed. Our findings point to the role of other factors in the pathogenesis of S-ECC, such as uncharacterized virulence genes, differences in gene expression and/or enzymatic activity, cooperation between S. mutans strains or with other members of the oral biota, and host factors.

Evidence-based clinical recommendations for the use of pit-and-fissure sealants: a report of the American Dental Association Council on Scientific Affairs.

This article presents evidence-based clinical recommendations for use of pit-and-fissure sealants developed by an expert panel convened by the American Dental Association (ADA) Council on Scientific Affairs. The panel addressed the following clinical questions. Under what circumstances should sealants be placed to prevent caries? Does placing sealants over early (noncavitated) lesions prevent progression of the lesion? Are there conditions that favor the placement of resin-based versus glass ionomer cement sealants in terms of retention or caries prevention? Are there any techniques that could improve sealants' retention and effectiveness in caries prevention? Staff of the ADA Division of Science conducted a MEDLINE search to identify systematic reviews and clinical studies published after the identified systematic reviews.

Genetic classification of severe early childhood caries by use of subtracted DNA fragments from Streptococcus mutans.

Streptococcus mutans is one of several members of the oral indigenous biota linked with severe early childhood caries (S-ECC). Because most humans harbor S. mutans, but not all manifest disease, it has been proposed that the strains of S. mutans associated with S-ECC are genetically distinct from those found in caries-free (CF) children. The objective of this study was to identify common DNA fragments from S. mutans present in S-ECC but not in CF children. Using suppressive subtractive hybridization, we found a number of DNA fragments (biomarkers) present in 88 to 95% of the S-ECC S. mutans strains but not in CF S. mutans strains. We then applied machine learning techniques including support vector machines and neural networks to identify the biomarkers with the most predictive power for disease status, achieving a 92% accurate classification of the strains as either S-ECC or CF associated. The presence of these gene fragments in 90 to 100% of the 26 S-ECC isolates tested suggested their possible functional role in the pathogenesis of S. mutans associated with dental caries.

Evidence-based clinical recommendations for the use of pit-and-fissure sealants: a report of the American Dental Association Council on Scientific Affairs.

BACKGROUND: This article presents evidence-based clinical recommendations for use of pit-and-fissure sealants developed by an expert panel convened by the American Dental Association Council on Scientific Affairs. The panel addressed the following clinical questions: Under what circumstances should sealants be placed to prevent caries? Does placing sealants over early (noncavitated) lesions prevent progression of the lesion? Are there conditions that favor the placement of resin-based versus. glass ionomer cement sealants in terms of retention or caries prevention? Are there any techniques that could improve sealants' retention and effectiveness in caries prevention? TYPES OF STUDIES REVIEWED: Staff of the ADA Division of Science conducted a MEDLINE search to identify systematic reviews and clinical studies published after the identified systematic reviews. At the panel's request, the ADA Division of Science staff conducted additional searches for clinical studies related to specific topics. The Centers for Disease Control and Prevention also provided unpublished systematic reviews that since have been accepted for publication. RESULTS: The expert panel developed clinical recommendations for each clinical question. The panel concluded that sealants are effective in caries prevention and that sealants can prevent the progression of early noncavitated carious lesions. CLINICAL IMPLICATIONS: These recommendations are presented as a resource to be considered in the clinical decision-making process. As part of the evidence-based approach to care, these clinical recommendations should be integrated with the practitioner's professional judgment and the patient's needs and preferences. The evidence indicates that sealants can be used effectively to prevent the initiation and progression of dental caries.

The effect of dental sealants on bacteria levels in caries lesions: a review of the evidence.

BACKGROUND: Concern about inadvertently sealing over caries often prevents dentists from providing dental sealants. The objective of the authors' review was to examine the effects of sealants on bacteria levels within caries lesions under dental sealants. METHODS: The authors searched electronic databases for comparative studies examining bacteria levels in sealed permanent teeth. To measure the effect of sealants on bacteria levels, they used the log(10) reduction in mean total viable bacteria counts (VBC) between sealed and not-sealed caries and the percentage reduction in the proportion of samples with viable bacteria. RESULTS: Six studies--three randomized controlled trials, two controlled trials and one before-and-after study-were included in the analysis. Although studies varied considerably, there were no findings of significant increases in bacteria under sealants. Sealing caries was associated with a 100-fold reduction in mean total VBC (four studies, 138 samples). Sealants reduced the probability of viable bacteria by about 50.0 percent (four studies, 117 samples). CONCLUSIONS: The authors found that sealants reduced bacteria in carious lesions, but that in some studies, low levels of bacteria persisted. These findings do not support reported concerns about poorer outcomes associated with inadvertently sealing caries. CLINICAL IMPLICATIONS: Practitioners should not be reluctant to provide sealants-an intervention proven to be highly effective in preventing caries-because of concerns about inadvertently sealing over caries.

Attitudes and expectations of treating deep caries: a PEARL Network survey.

A survey was conducted within a practice-based dental research network to determine dentists' treatment methods for deep caries lesions and whether the dentists' intended treatment approaches were influenced by their expectations for pulpal exposure. The survey further examined how general dentistry practices have adopted scientific evidence of caries classification, excavation, and capping techniques. Dentists were queried regarding liner use, hypersensitivity considerations, point of endodontic therapy, and anticipated vitality outcomes from Class I resin-based composite restorations over three to five years. Of the 93 practitioner-investigators who were in the network at the time of the survey, 85 (92%) completed it. Of those who responded, 62% said that they would remove all caries when presented with a case in which one would expect pulpal exposure, while 18% would partially remove caries and 21% would initiate endodontic treatment; 17% reported that they would utilize an antimicrobial agent before a liner or bonding agent during restoration. The outcomes projected for tooth vitality over the next three to five years were equivalent regardless of the caries removal approach or the use of a liner/bonding agent. When beginning the preparation, the method of treatment did not change if a pulpal exposure was anticipated, other than a threefold increase in immediate endodontic treatment. When dentists were given a direct pulp cap scenario, the projected use of a liner/bonding agent changed little while the vitality projections decreased. Overall survey findings indicate that approximately 20% of network dentists favor partial caries removal techniques and that deep caries treatment outcome studies are warranted, given the various treatments employed.

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples.

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.

Population structure of plasmid-containing strains of Streptococcus mutans, a member of the human indigenous biota.

There are suggestions that the phylogeny of Streptococcus mutans, a member of the human indigenous biota that is transmitted mostly mother to child, might parallel the evolutionary history of its human host. The relatedness and phylogeny of plasmid-containing strains of S. mutans were examined based on chromosomal DNA fingerprints (CDF), a hypervariable region (HVR) of a 5.6-kb plasmid, the rRNA gene intergenic spacer region (IGSR), serotypes, and the genotypes of mutacin I and II. Plasmid-containing strains were studied because their genetic diversity was twice as great as that of plasmid-free strains. The CDF of S. mutans from unrelated human hosts were unique, except those from Caucasians, which were essentially identical. The evolutionary history of the IGSR, with or without the serotype and mutacin characters, clearly delineated an Asian clade. Also, a continuous association with mutacin II could be reconstructed through an evolutionary lineage with the IGSR, but not for serotype e. DNA sequences from the HVR of the plasmid produced a well-resolved phylogeny that differed from the chromosomal phylogeny, indicating that the horizontal transfer of the plasmid may have occurred multiple times. The plasmid phylogeny was more congruent with serotype e than with mutacin II evolution, suggesting a possible functional correlation. Thus, the history of this three-tiered relationship between human, bacterium, and plasmid supported both coevolution and independent evolution.

Identification of unique bacterial gene segments from Streptococcus mutans with potential relevance to dental caries by subtraction DNA hybridization.

Using DNA subtractive hybridization, 49 unique gene segments were identified from a strain of Streptococcus mutans that was isolated from a patient with severe early childhood caries (S-ECC). Further hybridization with DNA from other S. mutans strains isolated from both caries-active and caries-free subjects yielded five unique sequences of DNA common to strains associated with S-ECC.

Dental caries: an infectious and transmissible disease where have we been and where are we going?

Dental caries is often referred to as an infectious disease, which means different things to different people. Pediatric dentists may be tempted to liken caries to other childhood diseases they encounter yet treat caries from mostly a mechanical or surgical approach. On the other end of the spectrum are clinicians who view the oral cavity as a site teaming with bacteria and think that reducing the overall microbial mass using antimicrobial agents will somehow affect this infectious disease. Here, we argue for another view, that is, to combine the most effective portion of each therapy to yield an effective outcome: the treatment and prevention of dental caries. Because primary teeth are eventually shed, pediatric dentists may be in an advantageous position to implement this approach since restorations to primary teeth need not be as durable as those made to permanent teeth. Moreover, pediatric dentists use sealants extensively and are used to working, for example, with rubber dams, especially in young patients. It may turn out that after more research is completed on the ontogeny of the oral microbial flora, changes or ecological shifts made in childhood become lifelong. More specifically, drastic reductions or eliminations of cariogenic bacteria, along with their sites for colonization in the primary dentition, may prevent or delay their transfer to the permanent dentition. Similarly, reductions in a mother's levels of cariogenic bacteria may delay or interrupt the transfer of cariogenic bacteria to her infant. Here, the goal is to discuss several concepts before their final inception into practices and to stimulate discussion of ideas, reframing the design to meet the demands of everyday practice. Ultimately, the proof will come from a large clinical trial conducted in many practices and patient populations.

Dental caries: an infectious and transmissible disease.

By definition, dental caries is an infectious and transmissible disease because it is caused by bacteria colonizing the tooth surfaces. Unlike most infectious diseases affecting humans, caries is the result of an imbalance of the indigenous oral biota rather than a nonindigenous, exogenous pathogen. The introduction of refined sugar into modern society's diet has tipped the balance from health to disease. New insight into the natural history of the leading cariogenic bacteria, the mutans streptococci, may contribute ways to control or prevent this infectious disease. Here, we use the host-parasite model as a platform for viewing the pathogenicity of the caries process in contrast to other infectious diseases.

Identification of Streptococcus sanguinis with a PCR-generated species-specific DNA probe.

The objective of the present study was to design a PCR-generated DNA probe and determine the specificity of the probe for the identification of clinical isolates of Streptococcus sanguinis. To do this, we examined over 200 arbitrarily primed PCR (AP-PCR) amplicon patterns obtained with DNA from clinical isolates of S. sanguinis. A 1.6-kb DNA amplicon that was common to all AP-PCR profiles was extracted from agarose gels and then cloned and sequenced. A search for a similar sequence in the GenBank database with the BLASTN program revealed that the 1.6-kb DNA fragment comprised an intergenic region between two housekeeping genes, uncC (proton-translocating ATPase) and murA (UDP-N-acetylglucosamine enolpyruvyl transferase). Three digoxigenin-labeled DNA probes were synthesized on the basis of the sequence of the 1.6-kb fragment: the sequence of probe SSA-1 contained the proton-translocating ATPase (uncC) and the entire intergenic region, the sequence of probe SSA-2 contained only the intergenic region, and the sequence of probe SSA-3 contained an internal region of the murA gene. Dot blot hybridization showed that the three probes displayed signals for hybridization to both S. sanguinis strain ATCC 10556 and the S. sanguinis clinical isolates. Probe SSA-1, however, hybridized to DNA from S. oralis and S. mitis. Probe SSA-3 hybridized to DNA from S. gordonii, S. mitis, S. oralis, S. parasanguinis, and S. vestibularis. The probe SSA-2-specific intergenic region appeared to be specific for S. sanguinis. The results from this study suggest that probe SSA-2 may serve as a species-specific DNA probe for the identification of clinical isolates of S. sanguinis.