Microbial Indicators of Dental Health, Dysbiosis, and Early Childhood Caries.

Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children (N = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) (n = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera Prevotella and Leptotrichia were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.

Subgingival Microbiota during Healthy Pregnancy and Pregnancy Gingivitis.

INTRODUCTION: Previous studies have largely explored the microbial composition and pathogenesis of pregnancy gingivitis. However, the patterns of microbial colonization during pregnancy in the absence of pregnancy gingivitis have rarely been studied. Characterization of the oral microbiome in pregnant women with healthy gingiva is an important initial step in understanding the role of the microbiome in progression to pregnancy gingivitis. OBJECTIVES: In this study, we compared the oral microbiome of pregnant women without gingivitis (healthy pregnancy) with pregnant women having gingivitis and nonpregnant healthy women to understand how pregnancy modifies the oral microbiome and induces progression to pregnancy gingivitis. METHODS: Subgingival plaque samples were collected from Chinese pregnant women with gingivitis (n = 10), healthy pregnant women (n = 10), and nonpregnant healthy women (n = 10). The Illumina MiSeq platform was used to perform 16S rRNA gene sequencing targeting the V4 region. RESULTS: The alpha and beta diversity was significantly different between pregnant and nonpregnant women, but minimal differences were observed between pregnant women with and without gingivitis. Interestingly, the oral bacterial community showed higher abundance of pathogenic taxa during healthy pregnancy as compared with nonpregnant women despite similar gingival and plaque index scores. However, when compared with overt pregnancy gingivitis, pathogenic taxa were less abundant during healthy pregnancy. PICRUSt analysis (phylogenetic investigation of communities by reconstruction of unobserved states) also suggested no difference in the functional capabilities of the microbiome during pregnancy, irrespective of gingival disease status. However, metabolic pathways related to amino acid metabolism were significantly increased in healthy pregnant women as compared with nonpregnant women. CONCLUSION: The presence of pathogenic taxa in healthy pregnancy and pregnancy gingivitis suggests that bacteria may be necessary for initiating disease development but progression to gingivitis may be influenced by the host environmental factors. More efforts are required to plan interventions aimed at sustaining health before the appearance of overt gingivitis. KNOWLEDGE TRANSFER STATEMENT: The results of this study draw attention to the importance of oral health maintenance during pregnancy, as women without any prenatal oral conditions are predisposed to the risk of developing pregnancy gingivitis. Hence, it is important to incorporate comprehensive assessment of oral health in the prenatal health care schedules. Pregnant woman should be screened for oral risks, counseled on proper oral hygiene and expected oral changes, and referred for dental treatment, when necessary.

Home sampling is a feasible method for oral microbiota analysis for infants and mothers.

OBJECTIVES: Large longitudinal cohort studies in infants are needed to understand oral microbiome maturation in relation to general health. The logistics of such studies are complex and costs involved high. Methods like home sampling by caretakers might be a solution to these issues. This study aimed to evaluate feasibility of home sampling by caretakers and to assess which oral niche provides the most reliable sample. METHODS: In this cross-sectional study 30 mothers and their infants aged 2-15 months participated. Swabs of the tongue, buccal mucosa, saliva, and dental plaque of the mother and the infant were collected by the mother after watching an instruction video. Thereafter, the trained researcher repeated the sample collection. Variations on the sampling protocol were listed. Bacterial DNA was quantified and microbial composition was assessed using 16S rDNA amplicon sequencing. RESULTS: None of the sampled niches appeared to be unfeasible based on interviews and observed variations on protocol. No significant differences in bacterial DNA concentration between operators (mother and researcher) were found. In infant's saliva, Shannon diversity of samples collected by the researcher was significantly higher than those collected by mothers (p = 0.0009) and the bacterial composition was influenced by variations on sampling protocol (p = 0.01). CONCLUSIONS: Home sampling by caretakers is a feasible method for oral sample collection in infants and mothers. Oral samples collected by mothers resemble samples collected by a trained researcher, with the tongue sample being the most similar and saliva the least. CLINICAL SIGNIFICANCE: Home sampling can simplify longitudinal oral microbiota collection.

Dental aerosols: microbial composition and spatial distribution.

Background: High-speed dental instruments produce aerosols, which can contribute to the transmission of pathogenic microorganisms. The aim of this study is to describe the microbial load and - composition and spatial distribution of aerosols in dental clinics. Methods: In four dental clinics active and passive sampling methods were used before, during and after treatment and at different locations. Retrieved colony forming units (CFU) were sequenced for taxon identification. Results: The samples contained up to 655 CFU/plate/30 minutes and 418 CFU/m3/30 minutes during dental treatment for active and passive sampling, respectively. The level of contamination after treatment and at 1.5 m distance from the patient's head was similar to the start of the day. The highest contamination was found at the patient's chest area. The aerosols consisted of 52 different taxa from human origin and 36 from water. Conclusion: Contamination in dental clinics due to aerosols is mainly low, although high level of contamination with taxa from both human and water origin was found within 80 cm around the head of the patient. Our results stress the importance of infection control measures on surfaces in close proximity to the head of the patient as well as in dental water lines.

Impact of the early-life skin microbiota on the development of canine atopic dermatitis in a high-risk breed birth cohort.

Canine atopic dermatitis (CAD) is a prevalent inflammatory skin disease of dogs worldwide. Certain breeds such as the West Highland White Terriers (WHWT) are predisposed to suffer from CAD. Microbial dysbiosis is known to play a significant role in the pathogenesis of the disease, which is similar to its human counterpart, atopic dermatitis (AD). To date, no large cohort-study has been conducted in a predisposed dog breed to study the impact of the early-life microbiota on the development of CAD, as well as the possible implication of factors such as hygiene and access to the outdoors. In this study skin samples of 143 WHWT, including 109 puppies up to three weeks old and 34 parent dogs, from 17 breeders, were subjected to 16S rRNA gene and ITS2 amplicon sequencing to disclose the bacterial and fungal oral and skin microbiota, respectively. The oral samples served as a control group to confirm differences between haired and mucosal surfaces. The cutaneous microbiota differed between sample sites and age of the dogs. The season of sampling, geographical origin as well as hygiene status of the household and the access to the outdoors shaped the skin microbiota of the puppies significantly. However, we found that the individual early-life microbiota did not predispose for the later development of CAD.

Maturation of the Oral Microbiome in Caries-Free Toddlers: A Longitudinal Study.

Understanding the development of the oral microbiota in healthy children is of great importance to oral and general health. However, limited data exist on a healthy maturation of the oral microbial ecosystem in children. Moreover, the data are biased by mislabeling "caries-free" populations. Therefore, we aimed to characterize the healthy salivary and dental plaque microbiome in young children. Caries-free (ICDAS [International Caries Detection and Assessment System] score 0) children (n = 119) and their primary caregivers were followed from 1 until 4 y of child age. Salivary and dental plaque samples were collected from the children at 3 time points (T1, ~1 y old; T2, ~2.5 y old; and T3, ~4 y old). Only saliva samples were collected from the caregivers. Bacterial V4 16S ribosomal DNA amplicons were sequenced using Illumina MiSeq. The reads were denoised and mapped to the zero-radius operational taxonomic units (zOTUs). Taxonomy was assigned using HOMD. The microbial profiles of children showed significant differences (P = 0.0001) over time. Various taxa increased, including Fusobacterium, Actinomyces, and Corynebacterium, while others showed significant decreases (e.g., Alloprevotella and Capnocytophaga) in their relative abundances over time. Microbial diversity and child-caregiver similarity increased most between 1 and 2.5 y of age while still not reaching the complexity of the caregivers at 4 y of age. The microbiome at 1 y of age differed the most from those at later time points. A single zOTU (Streptococcus) was present in all samples (n = 925) of the study. A large variation in the proportion of shared zOTUs was observed within an individual child over time (2% to 42% of zOTUs in saliva; 2.5% to 38% in dental plaque). These findings indicate that the oral ecosystem of caries-free toddlers is highly heterogeneous and dynamic with substantial changes in microbial composition over time and only few taxa persisting across the 3 y of the study. The salivary microbiome of 4-y-old children is still distinct from that of their caregivers.

Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study.

Routine postoperative antibiotic prophylaxis is not recommended for third molar extractions. However, amoxicillin still continues to be used customarily in several clinical practices worldwide to prevent infections. A prospective cohort study was conducted in cohorts who underwent third molar extractions with (group EA, n = 20) or without (group E, n = 20) amoxicillin (250 mg three times daily for 5 days). Further, a control group without amoxicillin and extractions (group C, n = 17) was included. Salivary samples were collected at baseline, 1-, 2-, 3-, 4-weeks and 3 months to assess the bacterial shift and antibiotic resistance gene changes employing 16S rRNA gene sequencing (Illumina-Miseq) and quantitative polymerase chain reaction. A further 6-month follow-up was performed for groups E and EA. Seven operational taxonomic units reported a significant change from baseline to 3 months for group EA (adjusted p < 0.05). No significant change in relative abundance of bacteria and ß-lactamase resistance genes (TEM-1) was observed over 6 months for any group (adjusted p > 0.05). In conclusion, the salivary microbiome is resilient to an antibiotic challenge by a low-dose regimen of amoxicillin. Further studies evaluating the effect of routinely used higher dose regimens of amoxicillin on gram-negative bacteria and antibiotic resistance genes are warranted.

Candida albicans alters the bacterial microbiome of early in vitro oral biofilms.

The yeast Candida albicans is an oral commensal microorganism, occurring in the oral cavity of 50-70% of healthy individuals. Its effect on oral ecology has mostly been studied using dual-species models, which disregards the complex nature of oral biofilms. The aim of this study was to culture C. albicans in a complex model to study its effect on oral biofilms. Biofilms, inoculated using pooled stimulated saliva with or without addition of C. albicans, were grown under anaerobic, aerobic, or aerobic +5% CO2 conditions. Red autofluorescence was quantified using a spectrophotometer and visualized in fluorescence photographs. The microbiome of 5 h biofilms was determined using 16S rDNA sequencing. C. albicans was only able to proliferate in biofilms grown under aerobic conditions. After 48 h, C. albicans did not induce differences in total biofilm formation, lactic acid accumulation (cariogenic phenotype) or protease activity (periodontitis phenotype). In vitro, anaerobically grown biofilms developed red autofluorescence, irrespective of inoculum. However, under aerobic conditions, only C. albicans-containing biofilms showed red autofluorescence. Facultative or strict anaerobic Veillonella, Prevotella, Leptotrichia, and Fusobacterium genera were significantly more abundant in biofilms with C. albicans. Biofilms without C. albicans contained more of the aerobic and facultative anaerobic genera Neisseria, Rothia, and Streptococcus. The presence of C. albicans alters the bacterial microbiome in early in vitro oral biofilms, resulting in the presence of strictly anaerobic bacteria under oxygen-rich conditions. This in vitro study illustrates that C. albicans should not be disregarded in healthy oral ecosystems, as it has the potential to influence bacteria significantly.

Microbial profiles at baseline and not the use of antibiotics determine the clinical outcome of the treatment of chronic periodontitis.

Antibiotics are often used in the treatment of chronic periodontitis, which is a major cause of tooth loss. However, evidence in favour of a microbial indication for the prescription of antibiotics is lacking, which may increase the risk of the possible indiscriminate use of antibiotics, and consequent, microbial resistance. Here, using an open-ended technique, we report the changes in the subgingival microbiome up to one year post-treatment of patients treated with basic periodontal therapy with or without antibiotics. Antibiotics resulted in a greater influence on the microbiome 3 months after therapy, but this difference disappeared at 6 months. Greater microbial diversity, specific taxa and certain microbial co-occurrences at baseline and not the use of antibiotics predicted better clinical treatment outcomes. Our results demonstrate the predictive value of specific subgingival bacterial profiles for the decision to prescribe antibiotics in the treatment of periodontitis, but they also indicate the need for alternative therapies based on ecological approaches.

The effect of propidium monoazide treatment on the measured bacterial composition of clinical samples after the use of a mouthwash.

OBJECTIVES: The use of an anti-microbial mouthwash results not only in a reduction of the number of viable cells in dental plaque but potentially also in a shift in the oral microbiome. DNA-based techniques may be appropriate to monitor these shifts, but these techniques amplify DNA from both dead and living cells. Propidium monoazide (PMA) has been used to overcome this problem, by preventing the amplification of DNA from membrane-damaged cells. The aim of this study was to evaluate the use of PMA when measuring compositional shifts in clinical samples after mouthwash use. MATERIALS AND METHODS: On two consecutive days, baseline samples from buccal surfaces, tongue, and saliva were obtained from six volunteers, after which they used a mouthwash (Meridol, GABA, Switzerland) twice daily for 14 days. Subsequently similar samples were obtained on two consecutive days. The microbial composition of the samples, with or without ex vivo PMA treatment, was assessed with 16S rRNA gene amplicon sequencing. RESULTS: Data showed a clear effect of mouthwash usage on the tongue and saliva samples. PMA treatment enhanced the observed differences only for the saliva samples. Mouthwash treatments did not affect the composition of the plaque samples irrespective of the use of PMA. CONCLUSION: The necessity to use a PMA treatment to block the DNA from dead cells in clinical studies aimed at measuring compositional shifts after the use of a mouthwash is limited to salivary samples. CLINICAL RELEVANCE: Measuring shifts in the oral microbiome could be hampered by the presence of DNA from dead cells.

Subcellular metabolic organization in the context of dynamic energy budget and biochemical systems theories.

The dynamic modelling of metabolic networks aims to describe the temporal evolution of metabolite concentrations in cells. This area has attracted increasing attention in recent years owing to the availability of high-throughput data and the general development of systems biology as a promising approach to study living organisms. Biochemical Systems Theory (BST) provides an accurate formalism to describe biological dynamic phenomena. However, knowledge about the molecular organization level, used in these models, is not enough to explain phenomena such as the driving forces of these metabolic networks. Dynamic Energy Budget (DEB) theory captures the quantitative aspects of the organization of metabolism at the organism level in a way that is non-species-specific. This imposes constraints on the sub-organismal organization that are not present in the bottom-up approach of systems biology. We use in vivo data of lactic acid bacteria under various conditions to compare some aspects of BST and DEB approaches. Due to the large number of parameters to be estimated in the BST model, we applied powerful parameter identification techniques. Both models fitted equally well, but the BST model employs more parameters. The DEB model uses similarities of processes under growth and no-growth conditions and under aerobic and anaerobic conditions, which reduce the number of parameters. This paper discusses some future directions for the integration of knowledge from these two rich and promising areas, working top-down and bottom-up simultaneously. This middle-out approach is expected to bring new ideas and insights to both areas in terms of describing how living organisms operate.

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

C. elegans DAF-12, Nuclear Hormone Receptors and human longevity and disease at old age.

In Caenorhabditis elegans, DAF-12 appears to be a decisive checkpoint for many life history traits including longevity. The daf-12 gene encodes a Nuclear Hormone Receptor (NHR) and is member of a superfamily that is abundantly represented throughout the animal kingdom, including humans. It is, however, unclear which of the human receptor representatives are most similar to DAF-12, and what their role is in determining human longevity and disease at old age. Using a sequence similarity search, we identified human NHRs similar to C. elegans DAF-12 and found that, based on sequence similarity, Liver X Receptor A and B are most similar to C. elegans DAF-12, followed by the Pregnane X Receptor, Vitamin D Receptor, Constitutive Andosteron Receptor and the Farnesoid X Receptor. Their biological functions include, amongst others, detoxification and immunomodulation. Both are processes that are involved in protecting the body from harmful environmental influences. Furthermore, the DAF-12 signalling systems seem to be functionally conserved and all six human NHRs have cholesterol derived compounds as their ligands. We conclude that the DAF-12 signalling system seems to be evolutionary conserved and that NHRs in man are critical for body homeostasis and survival. Genomic variations in these NHRs or their target genes are prime candidates for the regulation of human lifespan and disease at old age.

Two parameters account for the flocculated growth of microbes in biodegradation assays.

Microbes in activated sludge tanks mostly occur in flocs rather than in cell suspensions. Flocculation results in a limited supply of substrate to the bacteria inside the flocs, which reduces the biodegradation rate of organic compounds by several orders of magnitude. This article presents a simple two-parameter extension of growth models for cell suspensions to account for the ensuing reduction of the degradation rate. The additional parameters represent floc size at division and diffusion length. The biomass of small flocs initially increases exponentially at a rate equal to that of cell suspensions. After this first phase, the growth rate gradually decreases and finally the radius becomes a linear function of time. At this time flocs are large and have a kernel of dead biomass. This kernel arises when the substrate concentration decreases below the threshold level at which cells are just able to pay their maintenance costs. We deduce an explicit approximative expression for the interdivision time of flocs, and thereby for the growth of flocculated microbial biomass at constant substrate concentrations. The model reveals that the effect of stirring on degradation rates occurs through a reduction of the floc size at division. The results can be applied in realistic biodegradation quantifications in activated sludge tanks as long as substrate concentrations change slowly.