The Evolving Microbiome of Dental Caries.

Dental caries is a significant oral and public health problem worldwide, especially in low-income populations. The risk of dental caries increases with frequent intake of dietary carbohydrates, including sugars, leading to increased acidity and disruption of the symbiotic diverse and complex microbial community of health. Excess acid production leads to a dysbiotic shift in the bacterial biofilm composition, demineralization of tooth structure, and cavities. Highly acidic and acid-tolerant species associated with caries include Streptococcus mutans, Lactobacillus, Actinomyces, Bifidobacterium, and Scardovia species. The differences in microbiotas depend on tooth site, extent of carious lesions, and rate of disease progression. Metagenomics and metatranscriptomics not only reveal the structure and genetic potential of the caries-associated microbiome, but, more importantly, capture the genetic makeup of the metabolically active microbiome in lesion sites. Due to its multifactorial nature, caries has been difficult to prevent. The use of topical fluoride has had a significant impact on reducing caries in clinical settings, but the approach is costly; the results are less sustainable for high-caries-risk individuals, especially children. Developing treatment regimens that specifically target S. mutans and other acidogenic bacteria, such as using nanoparticles, show promise in altering the cariogenic microbiome, thereby combatting the disease.

Household, dietary, and clinical characteristics of childhood caries and overweight progression-A prospective cohort study.

BACKGROUND: Childhood caries and obesity are complex chronic diseases with negative health outcomes. AIM: This study sought a risk profile for childhood caries and overweight. DESIGN: Children were recruited into a longitudinal prospective cohort study. Caries and overweight characteristics were obtained at baseline, 6, 12, and 18 months. Sequential data modeling steps determined a disease risk profile. RESULTS: At baseline, 50% of the children (n = 194, 3.0 to 6.9 years) had caries; 24% were overweight, of whom 50% had caries. Correlation analysis separated child characteristics from household circumstances. Principal component modeling separated child snacking from meal-eating patterns, and household smoking from parent education variables. Baseline caries and overweight were not associated, but they grouped together in the modeling of composite features. Forty-five percent of children showed caries progression, 29% overweight progression, and 10% progression of both diseases. The strongest predictors of progression were disease presence, household-based characteristics, and sugary drinks. Children with caries and overweight progression shared multiple child- and household-based features. CONCLUSION: Individually, caries and overweight were not associated. Children with progression of both conditions shared a profile and multiple risk characteristics suggesting these findings could be useful in assessing the risk for the most extreme cases of caries and overweight.

Sugar Metabolism of Scardovia wiggsiae, a Novel Caries-Associated Bacterium.

Scardovia wiggsiae has been detected from caries in children and adolescents and has been suggested to be a caries-associated microorganism. To investigate the cariogenic potential of S. wiggsiae, we examined carbohydrate metabolism and acid productivity, the fluoride sensitivity of carbohydrate metabolism and the mechanism by which fluoride inhibits carbohydrate metabolism, and the acid sensitivity of carbohydrate metabolism in this bacterium. S. wiggsiae metabolized glucose and reduced the environmental pH to 3.5. It mainly produced acetic acid from glucose, together with small amounts of lactic and formic acid. The 50% inhibitory concentration of fluoride for acid production was 8.0 mM at pH 7.0 and 1.5 mM at pH 5.5, which were much higher than those of representative caries-associated bacteria, such as Streptococcus mutans. Metabolomic profiles showed the accumulation of 3-phosphoglycerate and a marked reduction in the pyruvate concentration in the presence of fluoride, suggesting that fluoride inhibits the latter half of glycolysis, including enolase activity. Enolase activity was inhibited by fluoride in S. wiggsiae, but it was more fluoride-tolerant than the enolase activity of S. mutans. Unlike in S. mutans, lactic acid did not inhibit acid production by S. wiggsiae at acidic pH. These results indicate that S. wiggsiae exhibits high acid production and tolerance to fluoride and lactic acid. S. wiggsiae possesses a unique metabolic pathway, the F6PPK shunt, which might allow it to avoid the lactate-formate pathway, including fluoride-sensitive enolase activity, and enable metabolic flow to the fluoride-tolerant acetate pathway. The fluoride tolerance of S. wiggsiae's enolase activity also increases the fluoride tolerance of its carbohydrate metabolism. The lactic acid tolerance of S. wiggsiae's acid production might result in S. wiggsiae having high acidogenic and aciduric potential and make it ecologically competitive in acidic environments, such as caries lesions, where lactic acid predominates.

Functional profiles of coronal and dentin caries in children.

Background: Dental caries results from a dysbiosis of tooth-associated biofilms and frequently extends through enamel into dentin which has a different structure and composition. Objective: To evaluate the metatranscriptome of caries to determine the metabolic potential of caries communities compared with health. Design: Samples from children, caries-free (CF: n = 4) or with coronal (CC: n = 5) or dentin (DC: n = 5) caries were examined for gene expression potential. Functional profiling was performed using HUMAnN2 (HMP Unified Metabolic Analysis Network). Results: There was increased gene expression diversity in DC compared with CC and CF. Genes in CF included alcohol dehydrogenase from Neisseria sicca, methylenetetrahydrofolate reductase from Streptococcus sanguinis and choline kinase from streptococci. Genes in CC mapped mainly to Streptococcus mutans. Arginine deiminase in DC mapped to S. sanguinis and Actinomyces naeslundii. Glycerol kinase genes mapped to S. sanguinis in all groups whereas glycerol kinase in DC were from Rothia, Prevotella and streptococci. Uracil-DNA glycosylase in DC mapped to Prevotella denticola and Actinomyces. Repressor LexA in DC mapped to Scardovia wiggsiae, Dialister invisus and Veillonella parvula. Conclusions: Functional profiling revealed enzyme activities in both caries and caries-free communities and clarified marked differences between coronal and dentin caries in bacterial composition and potential gene expression.

Scardovia wiggsiae and its potential role as a caries pathogen.

BACKGROUND: Streptococcus mutans has been strongly associated with dental caries but caries also occurs in its absence. Association of a new species, Scardovia wiggsiae with childhood caries suggests this could be a new caries pathogen. HIGHLIGHT: S. mutans is considered a caries pathogen based on its association with caries, and on its ability to produce acid, to survive low pH environments, and to induce caries in experimental animals. S. wiggsiae was significantly associated with severe-early childhood caries in the presence and absence of S. mutans. Further S. wiggsiae was elevated in initial carious lesions in adolescents with fixed orthodontic appliances. S. wiggsiae detection was enriched on a low pH agar suggesting acid-tolerance. S. wiggsiae isolates were acid tolerant and produced acid from several sugars at low initial pH values, and were not arginine deiminase positive, characteristics consistent with potential cariogenicity. Cariogenicity of S. wiggsiae was tested in a rat animal model in parallel with S. mutans. While S. wiggsiae by itself showed minimal caries induction, when co-inoculated with S. mutans, there was significant cavity production. CONCLUSION: S. wiggsiae was associated with advanced and initial caries, is acid tolerant and produces acid to low pH at initial neutral and low pH conditions. In combination with S. mutans, S. wiggsiae was detected in caries in an animal model. Together, these data suggest that S. wiggsiae has many of the characteristics consistent with its being a caries-associated species.

Quantitative assessment of salivary oral bacteria according to the severity of dental caries in childhood.

This study aimed to assess differences in selected oral bacteria in children according to the severity of dental caries. One hundred and thirty-six children, 36-60 months old were divided into three groups according to caries status: caries-free (CF) (n=47), early childhood caries (ECC) (n=40) and severe-early childhood caries (S-ECC) (n=49). Saliva was collected for detection and quantification of selected oral streptococci, Actinomyces naeslundii, Lactobacillus spp., Bifidobacterium spp., and Scardovia wiggsiae by quantitative-polymerase chain reaction. The results showed that the detection and quantitative levels of S. mutans, S. sobrinus, Bifidobacterium spp. and S. wiggsiae were significantly higher in S-ECC children compared to CF and ECC children, while for S. salivarius were significantly higher in CF compared to ECC and S-ECC children. There was no statistical difference among the clinical groups for S. mitis, S. oralis, A. naeslundii and Lactobacillus spp. levels and detection. S-ECC children had a lower monthly family income, started tooth brushing later and were breastfeed for a longer duration compared to CF children. S. mutans levels were positively correlated with S. wiggsiae and Bifidobacterium spp. levels, lower mother's education and child bottle-feeding before sleeping and negatively correlated with S. salivarius. It was concluded that in addition to S. mutans, other bacterial species, including bifidobacteria, Scardovia wiggsiae and S. sobrinus, are associated with severity of early childhood caries, although their role in the progress of dental caries remains unclear.

Oral microbial profiles of individuals with different levels of sugar intake.

The aim was to compare the oral microbial profiles in young adults with an intake of free sugars above or below the current recommendations by the WHO for sugar consumption. Seventy subjects completed a Quantitative Food Frequency Questionnaire to establish the proportion of free sugars in relation to the total energy intake (% E). Subjects with <5% E (n = 30) formed the low-sugar group, while those with ≥5% E (n = 40) were regarded as reference group. Saliva and plaque samples were analyzed by qPCR, and 52 of the plaque samples were assayed by HOMINGS. The HOMINGS analysis revealed a comparable core microbiota in plaque samples with Streptococcus, Leptotrichia, Actinobaculum, and Veillonella as predominant. No major differences between groups were revealed by α-diversity testing (p = 0.83), principal component analysis, or correspondence analysis. Higher relative abundance of Streptococcus sobrinus and Prevotella melaninogenica was observed in plaque samples in the reference group. By qPCR, Scardovia wiggsiae was associated with elevated sugar intake. The findings suggests that the amount of ingested sugars had a marginal influence on microbial profiles in dental plaque and saliva. However, some caries-associated species were less abundant in the dental plaque of the low sugar group.

Understanding Caries From the Oral Microbiome Perspective.

Dental caries is a major disease of the oral cavity with profound clinical significance. Caries results from a transition of a healthy oral microbiome into an acidogenic community of decreased microbial diversity in response to excessive dietary sugar intake. Microbiological cultivation, molecular identification, gene expression and metabolomic analyses show the importance of the entire microbial community in understanding the role of the microbiome in the pathology of caries.

In vitro Resistance Testing of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia to Triclosan.

AIM: To determine the sensitivity of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia to triclosan, and determine if these bacteria develop resistance to triclosan upon prolonged exposure. MATERIALS AND METHODS: Susceptibility to triclosan was tested against three periodontal pathogens P. gingivalis, P. intermedia, and T. forsythia. Escherichia coli strains sensitive and resistant to triclosan were used as biological controls to confirm the efficacy of triclosan in the assays. Agar plates were prepared locally with vitamin K and hemin-supplemented medium. RESULTS: Porphyromonas gingivalis and P. intermedia did not grow on plates containing ≥ 2 µg/ml triclosan, while T. forsythia did not grow on ≥ 1.66 µg/ml. Colonies of P. intermedia resistant to triclosan developed after prolonged incubation at 2 µg/ml, but this resistance disappeared during subculture in the absence of triclosan. CONCLUSION: No significant resistance to triclosan was detected for these species. CLINICAL SIGNIFICANCE: Dental products containing triclosan can be beneficial in controlling periodontal disease.

Antimicrobial peptides in saliva of children with severe early childhood caries.

OBJECTIVE: Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. DESIGN: 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n=29), ECC group (n=25) and S-ECC group (n=29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human ß-defensin 2 (hBD-2), human ß-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. RESULTS: Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. CONCLUSIONS: Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides.

Piracy on the molecular level: human herpesviruses manipulate cellular chemotaxis.

Cellular chemotaxis is important to tissue homeostasis and proper development. Human herpesvirus species influence cellular chemotaxis by regulating cellular chemokines and chemokine receptors. Herpesviruses also express various viral chemokines and chemokine receptors during infection. These changes to chemokine concentrations and receptor availability assist in the pathogenesis of herpesviruses and contribute to a variety of diseases and malignancies. By interfering with the positioning of host cells during herpesvirus infection, viral spread is assisted, latency can be established and the immune system is prevented from eradicating viral infection.

CREATING PEDIATRIC SPECIALTY AMBULATORY CARE DASHBOARDS.

Although interpretation is challenging, establishing dashboards for certain metrics does raise awareness of the importance of quality to patients, physicians and health care organization administrators.

Effect of Antimicrobial Interventions on the Oral Microbiota Associated with Early Childhood Caries.

PURPOSE: The purposes of this systematic literature review were to identify research-based evidence for an effect of antimicrobial therapeutic approaches on the cariogenic microbiota and early childhood caries (ECC) outcomes; and to review methods used to perform microbial assessments in clinical studies of ECC. METHODS: Multiple databases were searched; only clinical cohort studies and randomized controlled trials published from 1998 to 2014 were selected. A total of 471 titles and abstracts were identified; 114 studies met the inclusion criteria for a full review, from which 41 studies were included in the meta-analyses. RESULTS: In most of the reviewed studies, moderate reductions in cariogenic bacterial levels, mainly in mutans streptococci (MS), were demonstrated following the use of antimicrobial agents, but bacterial regrowth occurred and new carious lesions developed once the treatment had ceased, particularly in high-risk children. Relatively consistent findings suggested that anti-cariogenic microbial interventions in mothers significantly reduced MS acquisition by children. However, studies of the long-term benefits of ECC prevention are lacking. CONCLUSION: Based on the meta-analyses, antimicrobial interventions and treatments show temporary reductions in MS colonization levels. However, there is insufficient evidence to indicate that the approaches used produced sustainable effects on cariogenic microbial colonization or ECC reduction and prevention.

Development of human B cells and antibodies following human hematopoietic stem cell transplantation to Rag2(-/-)γc(-/-) mice.

Humanized mice represent a valuable model system to study the development and functionality of the human immune system. In the RAG-hu mouse model highly immunodeficient Rag2(-/-)γc(-/-) mice are transplanted with human CD34(+) hematopoietic stem cells, resulting in human hematopoiesis and a predominant production of B and T lymphocytes. Human adaptive immune responses have been detected towards a variety of antigens in humanized mice but both cellular and humoral immune responses tend to be weak and sporadically detected. The underlying mechanisms for inconsistent responses are poorly understood. Here, we analyzed the kinetics of human B cell development and antibody production in RAG-hu mice to better understand the lack of effective antibody responses. We found that T cell levels in blood did not significantly change from 8 to 28 weeks post-engraftment, while B cells reached a peak at 14 weeks. Concentrations of 3 antibody classes (IgM, IgG, IgA) were found to be at levels about 0.1% or less of normal human levels, but human antibodies were still detected up to 32 weeks after engraftment. Human IgM was detected in 92.5% of animals while IgG and IgA were detected in about half of animals. We performed flow cytometric analysis of human B cells in bone marrow, spleen, and blood to examine the presence of precursor B cells, immature B cells, naïve B cells, and plasma B cells. We detected high levels of surface IgM(+) B cells (immature and naïve B cells) and low levels of plasma B cells in these organs, suggesting that B cells do not mature properly in this model. Low levels of human T cells in the spleen were observed, and we suggest that the lack of T cell help may explain poor B cell development and antibody responses. We conclude that human B cells that develop in humanized mice do not receive the signals necessary to undergo class-switching or to secrete antibody effectively, and we discuss strategies to potentially overcome these barriers.

Pulp and plaque microbiotas of children with severe early childhood caries.

BACKGROUND AND OBJECTIVE: Bacterial invasion into pulps of primary teeth can lead to infection and premature tooth loss in children. This pilot study aimed to explore whether the microbiota of carious exposures of dental pulps resembles that of carious dentin or that of infected root canals. DESIGN: Children with severe early childhood caries were studied. Children were consented and extent of caries, plaque, and gingivitis measured. Bacteria were sampled from carious lesion biofilms and vital carious exposures of pulps, and processed by anaerobic culture. Isolates were characterized from partial sequences of the 16S rRNA gene and identified by comparison with taxa in the Human Oral Microbiome Database (http://www.HOMD.org). The microbiotas of carious lesions and dental pulps were compared using univariate and multivariate approaches. RESULTS: The microbiota of cariously exposed pulps was similar in composition to that of carious lesion biofilms except that fewer species/taxa were identified from pulps. The major taxa identified belonged to the phyla Firmicutes (mainly streptococci) and Actinobacteria (mainly Actinomyces species). Actinomyces and Selenomonas species were associated with carious lesions whereas Veillonella species, particularly Veillonella dispar was associated with pulps. Other bacteria detected in pulps included Streptococcus mutans, Parascardovia denticolens, Bifidobacterium longum, and several Lactobacillus and Actinomyces species. By principal, component analysis pulp microbiotas grouped together, whereas those in caries biofilms were widely dispersed. CONCLUSIONS: We conclude that the microbiota of cariously exposed vital primary pulps is composed of a subset of species associated with carious lesions. Vital primary pulps had a dominant Firmicutes and Actinobacteria microbiota which contrasts with reports of endodontic infections which can harbor a gram-negative microbiota. The microbiota of exposed primary pulps may provide insight into bacterial species at the forefront of caries invasion in dentinal lesions that can invade into the pulp and the nature of species that need suppressing for successful pulp therapy.

Anaerobic culture to detect periodontal and caries pathogens.

BACKGROUND: Anaerobic culture has been critical in our understanding of the oral microbiotas. HIGHLIGHT: Studies in advanced periodontitis in the 1970's revealed microbial complexes that associated with different clinical presentations. Taxonomy studies identified species newly-observed in periodontitis as Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter (Wolinella) rectus and other Campylobacter species, and Tannerella (Bacteroides) forsythia. Anaerobic culture of initial periodontitis showed overlap in the microbiota with gingivitis, and added Selenomonas noxia and Filifactor alocis as putative periodontal pathogens. Porphyromonas gingivalis and T. forsythia were found to be associated with initial periodontitis in adults. The dominant microbiota of dental caries differs from that of periodontitis. The major cariogenic species are acidogenic and acid tolerant species particularly Streptococcus mutans, and Lactobacillus and Bifidobacterium species. Anaerobic culture of severe early childhood caries revealed a widely diverse microbiota, comparable to that observed using cloning and sequencing. The PCR-based cloning approach, however, underestimated Actinobacteria compared with culture. Only a subset of the caries-associated microbiota was acid tolerant, with different segments of the microbiota cultured on blood agar compared to a low pH acid agar. While the major caries-associated species was S. mutans, a new species, Scardovia wiggsiae, was significantly associated with early childhood caries. Higher counts of S. wiggsiae were also observed in initial white spot carious lesions in adolescents. CONCLUSION: In periodontitis and dental caries, anaerobic culture studies of advanced disease provided a comprehensive analysis of the microbiota of these infections. Anaerobic culture highlighted the limitation of PCR with standard primers that underestimate detection of Actinobacteria.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

Modelling of human herpesvirus infections in humanized mice.

The human herpesviruses (HHVs) are remarkably successful human pathogens, with some members of the family successfully establishing infection in the vast majority of humans worldwide. Although many HHV infections result in asymptomatic infection or mild disease, there are rare cases of severe disease and death found with nearly every HHV. Many of the pathogenic mechanisms of these viruses are poorly understood, and in many cases, effective antiviral drugs are lacking. Only a single vaccine exists for the HHVs and researchers have been unable to develop treatments to cure the persistent infections associated with HHVs. A major hindrance to HHV research has been the lack of suitable animal models, with the notable exception of the herpes simplex viruses. One promising area for HHV research is the use of humanized mouse models, in which human cells or tissues are transplanted into immunodeficient mice. Current humanized mouse models mostly transplant human haematopoietic stem cells (HSCs), resulting in the production of a variety of human immune cells. Although all HHVs are thought to infect human immune cells, the beta- and gammaherpesviruses extensively infect and establish latency in these cells. Thus, mice humanized with HSCs hold great promise to study these herpesviruses. In this review, we provide a historical perspective on the use of both older and newer humanized mouse models to study HHV infections. The focus is on current developments in using humanized mice to study mechanisms of HHV-induced pathogenesis, human immune responses to HHVs and effectiveness of antiviral drugs.

Humanized mice as a model to study human hematopoietic stem cell transplantation.

Hematopoietic stem cell (HSC) transplantation has the potential to treat a variety of human diseases, including genetic deficiencies, immune disorders, and to restore immunity following cancer treatment. However, there are several obstacles that prevent effective HSC transplantation in humans. These include finding a matched donor, having a sufficient number of cells for the transplant, and the potency of the cells in the transplant. Ethical issues prevent effective research in humans that could provide insight into ways to overcome these obstacles. Highly immunodeficient mice can be transplanted with human HSCs and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. As such, humanized mice serve as a model for human HSC transplantation. A variety of conditions have been analyzed for their impact on HSC transplantation to produce humanized mice, including the type and source of cells used in the transplant, the number of cells transplanted, the expansion of cells with various protocols, and the route of introduction of cells into the mouse. In this review, we summarize what has been learned about HSC transplantation using humanized mice as a recipient model and we comment on how these models may be useful to future preclinical research to determine more effective ways to expand HSCs and to determine their repopulating potential in vivo.