Human Caries Research Articles

The Mechanisms and Application Value of Postbiotics in Caries Prevention and Management.

Dental caries, one of the most prevalent diseases globally, affects individuals throughout their lifetimes. Recently, researchers have increasingly focused on postbiotics for caries prevention. Postbiotics, comprising inanimate microorganisms and/or their components, confer health benefits to the host. Growing evidence suggests postbiotics' potential anticaries effects. Specifically, numerous postbiotics have demonstrated the ability to inhibit dental caries onset and progression by modulating oral flora microecology and reducing human caries susceptibility. This review elaborates on the current research regarding postbiotics' anticaries effects, highlights some studies' shortcomings, and innovatively proposes that postbiotics could potentially influence tooth development and salivary characteristics through epigenetic modifications. Furthermore, it anticipates postbiotics' future application in personalised caries treatment, given their multifaceted anticaries potential.

SALIVARY BIOMARKERS OF DENTAL CARIES - A REVIEW ARTICLE

Saliva is the complex mixture of fluids that surrounds the oral tissues, secreted by the major and minor salivary glands1. Human saliva not only lubricates the oral tissues, making oral functions such as speaking, eating, and swallowing possible, but also protects teeth and oral mucosal surfaces in different ways. Dental caries is recognized as a multi-factorial infectious disease caused by complex interactions among acid-producing bacteria, fermentable carbohydrates and many host factors including saliva. Saliva is easy to collect, to store, and can be obtained at low cost in sufficient quantities for analysis by a non-invasive method. Thus it has the potential to be used in the early detection and diagnosis of various oral diseases due to the abundant biomarkers present in saliva. Saliva biomarkers such as salivary flow, salivary pH, salivary proteins, microorganisms etc, are used for the prediction, diagnosis, prognosis and management of dental caries, as well as for evaluating the outcome of therapeutic regimens. This article aims to providea brief overview of salivary biomarkers present which can be used for the early detection nd prevention of dental caries in humans.

Polyketides/nonribosomal peptides from Streptococcus mutans and their ecological roles in dental biofilm.

Streptococcus mutans is the major etiological agent of dental caries in humans. S. mutans overgrowth within dental biofilms can trigger biofilm dysbiosis, ultimately leading to the initiation or progression of dental caries. Polyketides and nonribosomal peptides (PKs/NRPs) are secondary metabolites with complex structures encoded by a cluster of biosynthetic genes. Although not essential for microbial growth, PKs/NRPs play important roles in physiological regulation. Three main classes of hybrid PKs/NRPs in S. mutans have been identified, including mutanobactin, mutanocyclin, and mutanofactin, encoded by the mub, muc, and muf gene clusters, respectively. These three hybrid PKs/NRPs play important roles in environmental adaptation, biofilm formation, and interspecies competition of S. mutans. In this review, we provide an overview of the major hybrid PKs/NRPs of S. mutans, including mutanobactin, mutanocyclin, and mutanofactin and address their ecological roles in dental biofilms. We place specific emphasis on important questions that are yet to be answered to provide novel insights into the cariogenic mechanism of S. mutans and facilitate improved management of dental caries. We highlight that S. mutans PKs/NRPs may be potential novel targets for the prevention and treatment of S. mutans-induced dental caries. The development of genomics, metabolomics, and mass spectrometry, together with the integration of various databases and bioinformatics tools, will allow the identification and synthesis of other secondary metabolites. Elucidating their physicochemical properties and their ecological roles in oral biofilms is crucial in the identification of novel targets for the ecological management of dental caries.

A scoping review on the association between early childhood caries and life on land: The Sustainable Development Goal 15.

The Sustainable Development Goal 15 (SDG15) deals with protecting, restoring, and promoting the sustainable use of terrestrial ecosystems, sustainably managing forests, halting and reversing land degradation, combating desertification and halting biodiversity loss. The purpose of this scoping review was to map the current evidence on the association between SDG 15 and Early Childhood Caries (ECC). This scoping review was reported in accordance with the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (PRISMA-ScR) guidelines. Formal literature searches were conducted in PubMed, Web of Science, and Scopus in March 2023 using key search terms. Studies with the criteria (in English, with full text available, addressing component of life on land, focusing on dental caries in humans, with results that can be extrapolated to control ECC in children less than 6 years of age) were included. Retrieved papers were summarised and a conceptual framework developed regarding the postulated link between SDG15 and ECC. Two publications met the inclusion criteria. Both publications were ecological studies relating environmental findings to aggregated health data at the area level. One study concluded that the eco-hydrogeological environment was associated with human health, including caries. The other reported that excessive calcium was associated with the presence of compounds increasing groundwater acidity that had an impact on human health, including caries. The two ecological studies were linked to SDG 15.1. It is also plausible that SDG 15.2 and SDG 15.3 may reduce the risk for food insecurity, unemployment, gender inequality, zoonotic infections, conflict and migration; while SDG 15.4 may improve access to medicinal plants such as anticariogenic chewing sticks and reduction in the consumption of cariogenic diets. There are currently no studies to support an association between ECC and SDG15 although there are multiple plausible pathways for such an association that can be explored. There is also the possibility of synergistic actions between the elements of soil, water and air in ways that differentially affect the risk of ECC. Studies on the direct link between the SDG15 and ECC are needed. These studies will require the use of innovative research approaches.

Antimicrobial Peptides Targeting Streptococcus mutans: Current Research on Design, Screening and Efficacy.

Antimicrobial peptides (AMPs) are small-molecule peptides that play a vital role in the nonspecific immune defense system of organisms. They mainly kill microorganisms by physically destroying the cell membrane and causing the leakage of contents. AMPs have attracted much attention as potential alternatives to antibiotics due to their low susceptibility to resistance. Streptococcus mutans (S. mutans) is one of the main causative agents of human dental caries. The design, screening, and efficacy evaluation of AMPs targeting S. mutans offer new possibilities for the prevention and treatment of oral diseases, especially dental caries, in the future. This article reviews AMPs from different sources that have inhibitory effects on S. mutans, discusses the mechanism of action of AMPs against S. mutans biofilms, and focuses on the research progress of screening methods, design modification, and biological activity evaluation of AMPs. We hope to provide insights and reference value for the development of new biologics.

A scoping review on the links between sustainable development goal 14 and early childhood caries

BackgroundThe Sustainable Development Goal (SDG) 14 addresses life below the waters, an important source of protein and contributor to global food security and economic development. Our aim was to explore possible evidence on the links between life below water and early childhood caries (ECC).MethodsThis scoping review identified articles on the link between life below water and caries according to the PRISMA-ScR guidelines. Three electronic databases (PubMed, Web of Science, and Scopus) were systematically searched in January 2023, using specific search terms. Studies written in English, with full text available, addressing life under water, focusing on dental caries in humans, with results that can be extrapolated to control ECC in children less than 6 years of age were included in the review. Descriptive statistics were used to summarize the retrieved papers and graphical presentation was used for visualization.ResultsThere were 224 publications retrieved of which 13 studies, published between 1960 and 2022, were included in the analysis. The papers originated from Asia (7/13), North America (3/13), Europe (1/13), and 2/13 had multi-country authorship. Also, four laboratory studies extracted agents from marine products to determine their efficacy in preventing caries formation and preventing/slowing plaque formation; four letters discussed the caries prevention potential of sea salt as a source of fluoride; and two review articles about the positive effects of extracted marine products for caries prevention. Most (11/13) studies addressed target 14.1 concerned with enriching the marine environment with nutrients and minerals; two addressed target 14.4 focused on ensuring fish stocks are within biologically sustainable levels; two addressed target 14.7 aimed at increasing the economic benefits through sustainable use of marine resources such as fisheries; and one focused on target 14.5 aimed at conserving marine areas by increasing protected areas. In addition, one ecological study assessed the association between the ecosystem and ECC.ConclusionsCurrently, there is little known about the impact of protection of marine and coastal ecosystem from pollution and ocean acidification on the risk of ECC. Further evidence on possible associations between life below water and ECC management is needed.

Catalase produced by Candida albicans protects Streptococcus mutans from H2O2 stress-one more piece in the cross-kingdom synergism puzzle.

Co-infection with Streptococcus mutans and Candida albicans is associated with dental caries, and their co-cultivation results in enhanced biofilm matrix production that contributes to increased virulence and caries risk. Moreover, the catalase-negative S. mutans demonstrates increased oxidative stress tolerance when co-cultivated in biofilms with C. albicans, a catalase-producing yeast. Here, we sought to obtain mechanistic insights into the increased H2O2 tolerance of S. mutans when co-cultivated with clinical isolates of Candida glabrata, Candida tropicalis, and C. albicans. Additionally, the C. albicans SC5314 laboratory strain, its catalase mutant (SC5314Δcat1), and S. mutans UA159 and its glucosyltransferase B/C mutant (UA159ΔgtfB/C) were grown as single- and dual-species biofilms. Time-kill assays revealed that upon acute H2O2 challenge, the survival rates of S. mutans in dual-species biofilms with the clinical isolates and C. albicans SC5314 were greater than when paired with SC5314Δcat1 or as a single-species biofilm. Importantly, this protection was independent of glucan production through S. mutans GtfB/C. Transwell assays and treatment with H2O2-pre-stimulated C. albicans SC5314 supernatant revealed that this protection is contact-dependent. Biofilm stability assays with sublethal H2O2 or peroxigenic Streptococcus A12 challenge resulted in biomass reduction of single-species S. mutans UA159 and dual-species with SC5314Δcat1 biofilms compared to UA159 biofilms co-cultured with C. albicans SC5314. S. mutans oxidative stress genes were upregulated in single-species biofilms when exposed to H2O2, but not when S. mutans was co-cultivated with C. albicans SC5314. Here, we uncovered a novel, contact-dependent, synergistic interaction in which the catalase of C. albicans protects S. mutans against H2O2. IMPORTANCE It is well established that co-infection with the gram-positive caries-associated bacterium Streptococcus mutans and the yeast pathobiont Candida albicans results in aggressive forms of caries in humans and animal models. Together, these microorganisms form robust biofilms through enhanced production of extracellular polysaccharide matrix. Further, co-habitation in a biofilm community appears to enhance these microbes' tolerance to environmental stressors. Here, we show that catalase produced by C. albicans protects S. mutans from H2O2 stress in a biofilm matrix-independent manner. Our findings uncovered a novel synergistic trait between these two microorganisms that could be further exploited for dental caries prevention and control.

Assessment of the Periodontal Cementum Ablation Depth during Root Planing by an Er:YAG Laser at Different Energy Densities: An Ex Vivo Study.

An important and non-adapted delivered energy of Er:YAG laser can eliminate the total thickness of root cementum during root planing. Conversely, the preservation of a partial layer of cementum covering the roots is vital for any periodontal ligament regeneration. Thus, the assessment of the cementum ablation depth produced by each energy density of Er:YAG laser is essential before considering its use for the periodontal planing and treatment of the cementum and root surfaces. Assessment of the cementum ablation depth at different energy densities of the Er:YAG laser is the aim of this study. A total of 48 human caries free molars were collected and used in this study. Areas to be irradiated were delimited by two longitudinal grooves (0.5 mm depth). Roots were divided randomly into four groups (4 × n = 12). An Er:YAG laser (2.94 µm) was used with a side-firing tip (R600T) with a 600 µm diameter and a frequency of 20 Hz combined with a cooling system of air 6 mL/min and water 4 mL/min. We used a super short pulse mode (SSP: pulse duration: 50 μs). We used a single irradiation passage backward from apex to cervical parts at 1 mm/s with a slight contact and at an angle of 15° to 30° between the tip and the root surface. Different energies were selected: 30 mJ, 40 mJ, 50 mJ, and 60 mJ. Microscopic observations showed that the average of the ablation depth increased with the increase of the delivered energy from 30 mJ to 60 mJ. Mean values of the ablation depths were respectively as follows: 43.75 ± 4.89 µm for the energy of 30 mJ, 50.05 ± 3.72 µm for 40 mJ, 65.56 ± 10.35 µm for 50 mJ, and 74.80 ± 15.23 µm for 60 mJ. A statistically significant difference existed between the ablation depth of all groups. Based on our results, the depth of cementum debridement is related to the level of the delivered energy. The lowest energy levels (30 mJ and 40 mJ) can ablate the root cementum surface for a variable depth from 43.75 ± 4.89 μm to 50.05 ± 3.72 μm.

Diarylureas: New Promising Small Molecules against Streptococcus mutans for the Treatment of Dental Caries.

Dental caries is a biofilm-mediated disease that represents a worldwide oral health issue. Streptococcus mutans has been ascertained as the main cariogenic pathogen responsible for human dental caries, with a high ability to form biofilms, regulated by the quorum sensing. Diarylureas represent a class of organic compounds that show numerous biological activities, including the antimicrobial one. Two small molecules belonging to this class, specifically to diphenylureas, BPU (1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea) and DMTU (1,3-di-m-tolyl-urea), showed interesting results in studies regarding the antimicrobial activity against the cariogenic bacterium S. mutans. Since there are not many antimicrobials used for the prevention and treatment of caries, further studies on these two interesting compounds and other diarylureas against S. mutans may be useful to design new effective agents for the treatment of caries with generally low cytotoxicity.

Evaluation of Streptocin SH3, a Bacteriocin produced by Streptococcus sanguinis isolated from Human Dental Plaque

Background: Bacteriocin is a peptidic toxin has many advantages to bacteria in their ecological niche and has strong antibacterial activity. Objective: The aim of this study was to evaluation of bacteriocin using Streptococcus sanguinis isolated from human dental caries. Subjects and Methods: Thirty five streptococcus isolates were diagnosed and tested for their production of bacteriocin, and then the optimal conditions for production of bacteriocin were determined. After that, the purification of bacteriocin was made partially by ammonium sulfate at 95% saturation levels, followed by and gel filtration chromatography using Sephadex G-50 column. Finally, physical characteristics were also studied and determined the bacteriocin stability. Results: Among all streptococcal isolates, the S. sanguinis SH3 isolate with batter antimicrobial activity was selected, and used in further experiments. The optimum conditions for bacteriocin production were in Todd Hewitt broth (THB) supplemented with 1% glucose at 37°C for 24h under anaerobic conditions and inoculum size of (1.2 ˟ 109CFU/ml) and pH 7, where the inhibition zone diameter reached to 29 mm against Enterococcus faecalis. The purified bacteriocin had high inhibitory activity against S. pneumoniae, S. pyogenes, E. faecalis, E. faecium and Leuconostoc mesenteroides. Streptocin SH3 keeps its effectiveness within the pH range 3-10, and temperature until 80ºC for 20 min. Conclusion: Bacteriocin produced from S. sanguinis has high activity against many pathogens and has high stability over wide ranges of temperature and pH, which makes it a good alternative in the medical and food preservation fields

Streptococcus mutans Associated with Endo-Periodontal Lesions in Intact Teeth

A massive periodontal destruction can affect the root canal (RC) system and potentially expose the pulpo-dentinal complex to opportunistic bacteria. Streptococcus mutans is a major pathogen of human caries and periodontal diseases, and its virulence mostly resides in the ability to adhere to collagen and form biofilms, due to collagen-binding proteins (CBPs) Cnm and Cbm. Seventeen patients affected by severe endo-periodontal lesions without caries and/or exposure of pulpal tissue were subjected to tooth extraction and samples for microbiological investigation were collected from the root surface (RS) and RC. The collected swabs were cultivated and subjected to the quantitative real-time PCR (qPCR) for the detection of S. mutans and to the PCR for the cnm/cbm genes investigation, followed by next-generation sequencing (NGS). S. mutans DNA was detected in 12/17 (70.5%) RS samples and in 8/17 (47.0%) RC samples. In the CBPs screening of positive samples, the cnm gene was detected in 4/12 (33.3%) RS and in 1/8 (12.5%) RC samples, whilst all the samples tested negative for the cbm gene. The presence of the cnm gene could enhance the local virulence of the pathogens. Therefore, S. mutans have to be included as potential periodontopathogen bacterium in severe or refractory forms of periodontal diseases.

Nanoscale correlative X-ray spectroscopy and ptychography of carious dental enamel

This study reports the characterisation of human dental enamel caries using synchrotron nanoscale correlative ptychography and spectroscopic mapping in combination with scanning electron microscopy. A lamella ̴2.4 µm thick was extracted from a carious enamel region of a tooth using focused ion beam-scanning electron microscopy and transferred to two synchrotron beamlines to perform hard X-ray nano-fluorescence spectroscopy simultaneously with differential phase contrast mapping at a beam size of 55 × 45 nm. Soft X-ray ptychography data was then reconstructed with a pixel size of 8 nm. The two dimensional variation in chemistry and structure of carious enamel was revealed at the nanoscale, namely, the organisation of hydroxyapatite nano-crystals within enamel rods was imaged together with the inter-rod region. Correlative use of electron and X-ray scanning microscopies for the same sample allowed visualisation of the connection between structure and composition as presented in a compound image where colour indicates the relative calcium concentration in the sample, as indicated by the calcium Kα fluorescence intensity and grey scale shows the nanostructure. This highlights the importance of advanced correlative imaging to investigate the complex structure-composition relationships in nanomaterials of natural or artificial origin.

A novel universal adhesive for improved dentin remineralization with antibiofilm potential against Streptococcus mutans and other cariogenic-pathogens

Dental demineralization is a widespread problem and the main responsible for dental caries and dentin hypersensitivity. Dental caries is a biofilm-driven bacterial infection that causes mineral depletion in enamel and is associated with an increased prevalence of medical rehabilitative polymeric implants. Since existing tissue-engineering methods are incapable of manufacturing synthetic dentin adhesive, modern therapeutic interventions address the remineralization of caries-infected dentin to prevent its inevitable removal. In recent years, magnetic nanoparticle-coated adhesives were known to deliver drugs to the pulp and get better adhesive penetration into dentin. In the present study, we prepared and assessed the effectiveness of antimicrobial peptide (cecropin A) coated Fe3O4-nanoparticle on dentin remineralization, bond strength, and its antibiofilm potential against Streptococcus mutans, is associated with dental caries in humans. Prior to the experiment, the synthesized [email protected]3O4 nanoparticle was characterized and assessed its antibiofilm potential against S. mutans. The transcriptomic approach with qPCR analysis stated that [email protected]3O4 nanoparticle significantly downregulated the expression of biofilm-regulated genes such as GtfB, GtfC, GtfD, VicR, ComDE, and Smu0630 in S. mutans, which validates the outcome of in vitro results. Interestingly, the prepared [email protected]3O4-nanoparticle coated adhesive effectively increased the dentin remineralization and bond strength. Fascinatingly, [email protected]3O4 adhesive significantly hindered the biofilm formation of S. mutans on the dentin surface. Notably, the [email protected]3O4 adhesive accomplished reduced biofilm CFU count, lactic acid production, and biofilm metabolic activity. In addition, the [email protected]3O4 adhesive also reduced the biofilm formation of other caries-associated bacterial pathogens, including S. sobrinus, S. sanguinis, and S. mitis. Overall, the current research found that the [email protected]3O4 adhesive was more effective in remineralizing dentin lesions with antibiofilm potential in order to avoid Streptococcus-derived dental caries.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

ABSTRACTThe number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross β-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms.IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

Identification of the antibacterial action mechanism of diterpenoids through transcriptome profiling.

Effective antibacterial substances of Aralia continentalis have anti-biofilm and bactericidal activity to the oral pathogen Streptococcus mutans. In this study, three compounds extracted from A. continentalis were identified as acanthoic acid, continentalic acid, and kaurenoic acid by NMR and were further investigated how these diterpenoids affect the physiology of the S. mutans. When S. mutans was exposed to individual or mixed fraction of diterpenoids, severe growth defects and unique morphology were observed. The proportion of unsaturated fatty acids in the cell membrane was increased compared to that of saturated fatty acids in the presence of diterpenoids. Genome-wide gene expression profiles with RNA-seq were compared to reveal the mode of action of diterpenoids. Streptococcus mutans commonly enhanced the expression of 176 genes in the presence of the individual diterpenoids, whereas the expression of 232 genes was considerably reduced. The diterpenoid treatment modulated the expression of genes or operon(s) involved in cell membrane synthesis, cell division, and carbohydrate metabolism of S. mutans. Collectively, these findings provide novel insights into the antibacterial effect of diterpenoids to control S. mutans infection, which causes human dental caries.

Natural products’ potential to maintain/ameliorate oral health: A review

Many diseases such as human dental caries result in the destruction of tooth structure; dental caries is an infectious disease leading to the destruction of tooth structure due to the acids produced on the fermentation of dietary carbohydrates by acidogenic bacteria. Bacteria colonise non-shedding oral surfaces, and produce lactic, acetic, and formic acids. Preserving tooth structure using fluoride in dental products may have its adverse effects on human health. The use of fluoride-containing dental products without supervision in children when the teeth are developing can lead to fluorosis. Therefore, the main aim of the present review was to identify natural oral healthcare products having minimum or no adverse effects in maintaining the integrity of tooth enamel. The active compounds observed in the natural plant products till date are polyphenolic compounds which contain antibacterial effects, and a potential to shift demineralisation to remineralisation. But their mechanism of action remain unclear. Therefore, further studies are needed to determine the effectiveness of these natural products and enhance their effect.

Lactobacilli and human dental caries: more than mechanical retention.

Lactobacilli have been considered as major contributors to human dental caries for over a century. Recent in vitro model studies have shown that when compared to Streptococcus mutans, a keystone pathogen of human dental caries, the ability of lactobacilli to form biofilms is poor, although differences exist between the different major species. Further studies using molecular and bioinformatics approaches provide evidence that multiple mechanisms, including adhesin-receptor mediated physical contact with S. mutans, facilitate the adherence and establishment of lactobacilli on the tooth surface. There is also evidence that under conditions like continuous sugar consumption, weak acids and other antimicrobials such as bacteriocins from lactobacilli can become detrimental to the microbial community, especially those in the proximity. Details on the underlying mechanisms of how different Lactobacillus sp. establish and persist in the highly complex microbiota on the tooth surface await further investigation.

Hierarchical 2D to 3D micro/nano-histology of human dental caries lesions using light, X-ray and electron microscopy

Dental caries is a widespread disease that proceeds by damaging superficial tooth enamel by heterogeneous dissolution. Conventional histology identifies different zones within carious lesions by their optical appearance, but fails to quantify the underlying nanoscale structural changes as a function of specific location, impeding better understanding of the demineralisation process. We employ detailed collocative analysis using different imaging modalities, resolutions and fields of view. Focused ion beam-scanning electron microscopy (FIB-SEM) reveals subsurface 3D nanostructure within milled micro-sized volumes, whilst X-ray tomography allows minimally destructive 3D imaging over large volumes. Correlative combination of these techniques reveals fine detail of enamel rods, inter-rod substance, sheaths, crystallites and voids as a function of location. The degree of enamel demineralisation within the body of the lesion, near its front, and at the surface is visualized and quantified in 3D. We thus establish the paradigm of dental 3D nano-histology as an advanced platform for quantitative evaluation of caries-induced structural modification.

The fruB Gene of Streptococcus mutans Encodes an Endo-Levanase That Enhances Growth on Levan and Influences Global Gene Expression.

ABSTRACTStreptococcus mutans, the primary etiologic agent of human dental caries, and a variety of oral Streptococcus and Actinomyces spp. synthesize high molecular mass homopolymers of fructose (fructans) with predominantly β2,1- (inulins) or β2,6-linkages (levans). The ability of S. mutans to degrade fructans contributes to the severity of dental caries. The extracellular product of fruA of S. mutans is an exo- β-d-fructofuranosidase that releases fructose from levan and inulin. Located 70 bp downstream of fruA, fruB encodes a member of the glycoside hydrolase family 32, but the function of FruB has not been established. Growth assays performed using wild-type UA159 and fruB-deficient derivatives, with fructans as the sole carbohydrate source, showed a significant reduction in the growth rate of a fruB mutant on levan, but not on inulin. A purified, recombinant FruB protein degraded levan to release mainly fructooligosaccharides. Driven by the fruA promoter and a secondary promoter located in the 3′ region of the fruA sequence, the fruB gene is inducible by fructose and especially by levan, but a stable stem-loop structure in the intergenic region likely modulates transcriptional read-through from fruA. Transcriptomic analysis of UA159 and a fruB mutant grown on 0.2% levan revealed differential expression of genes encoding ABC transporters, transcriptional regulators and genes involved in growth and stress tolerance. The ability of FruB to enhance levan metabolism and the high degree of conservation of FruB across S. mutans isolates imply a significant contribution of FruB to the fitness and virulence of this pathogen in human dental biofilms.IMPORTANCE Carbohydrate metabolism and acid production are essential for the development of dental caries. As a by-product of sucrose metabolism, formation, and degradation of fructans enhances the severity of caries by S. mutans in animal models. This study highlights a significant breakthrough in identifying FruB in S. mutans as an endolevanase that contributes to efficient utilization of levan, a specific type of fructan produced by certain commensals but not S. mutans. Transcriptomic analysis revealed that FruB-dependent levan metabolism impacted global gene regulation, including a large number of novel genes. Considering the preference for levan by both FruA and FruB, the conservation of fruAB in S. mutans might represent a competitive advantage in access to the energy storage produced by dental microbiome. This is the first report demonstrating the presence of an endolevanase in S. mutans, therefore should be of broad interest to the fields of dental caries and complex carbohydrate metabolism.

Tolerable upper intake level for dietary sugars.

Following a request from five European Nordic countries, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was tasked to provide scientific advice on a tolerable upper intake level (UL) or a safe level of intake for dietary (total/added/free) sugars based on available data on chronic metabolic diseases, pregnancy‐related endpoints and dental caries. Specific sugar types (fructose) and sources of sugars were also addressed. The intake of dietary sugars is a well‐established hazard in relation to dental caries in humans. Based on a systematic review of the literature, prospective cohort studies do not support a positive relationship between the intake of dietary sugars, in isocaloric exchange with other macronutrients, and any of the chronic metabolic diseases or pregnancy‐related endpoints assessed. Based on randomised control trials on surrogate disease endpoints, there is evidence for a positive and causal relationship between the intake of added/free sugars and risk of some chronic metabolic diseases: The level of certainty is moderate for obesity and dyslipidaemia (> 50–75% probability), low for non‐alcoholic fatty liver disease and type 2 diabetes (> 15–50% probability) and very low for hypertension (0–15% probability). Health effects of added vs. free sugars could not be compared. A level of sugars intake at which the risk of dental caries/chronic metabolic diseases is not increased could not be identified over the range of observed intakes, and thus, a UL or a safe level of intake could not be set. Based on available data and related uncertainties, the intake of added and free sugars should be as low as possible in the context of a nutritionally adequate diet. Decreasing the intake of added and free sugars would decrease the intake of total sugars to a similar extent. This opinion can assist EU Member States in setting national goals/recommendations.