Candida virulence and ethanol-derived acetaldehyde production in oral cancer and non-cancer subjects.

OBJECTIVES: To compare biofilm-forming ability, hydrolytic enzymes and ethanol-derived acetaldehyde production of oral Candida isolated from the patients with oral cancer and matched non-oral cancer. MATERIAL AND METHODS: Fungal biofilms were grown in RPMI-1640 medium, and biofilm mass and biofilm activity were assessed using crystal violet staining and XTT salt reduction assays, respectively. Phospholipase, proteinase, and esterase production were measured using agar plate method, while fungal acetaldehyde production was assessed via gas chromatography. RESULTS: Candida isolated from patients with oral cancer demonstrated significantly higher biofilm mass (P = 0.031), biofilm metabolic activity (P < 0.001), phospholipase (P = 0.002), and proteinase (P = 0.0159) activity than isolates from patients with non-oral cancer. High ethanol-derived acetaldehyde-producing Candida were more prevalent in patients with oral cancer than non-oral cancer (P = 0.01). In univariate regression analysis, high biofilm mass (P = 0.03) and biofilm metabolic activity (P < 0.001), high phospholipase (P = 0.003), and acetaldehyde production ability (0.01) were significant risk factors for oral cancer; while in the multivariate regression analysis, high biofilm activity (0.01) and phospholipase (P = 0.01) were significantly positive influencing factors on oral cancer. CONCLUSION: These data suggest a significant positive association between the ability of Candida isolates to form biofilms, to produce hydrolytic enzymes, and to metabolize alcohol to acetaldehyde with their ability to promote oral cancer development.

Sterilization of rotary NiTi instruments within endodontic sponges.

AIM: To determine whether the following can be sterilized by autoclaving - endodontic sponges, rotary nickel-titanium (NiTi) instruments within endodontic sponges, and rotary NiTi instruments with rubber stoppers. METHODOLOGY: Sixty-four samples of eight different endodontic sponges (n = 512) were placed into brain heart infusion broth (BHI) for 72 h. An aliquot of this was then spread onto horse blood agar and cultured aerobically and anaerobically to test sterility at purchase. Bacterial suspensions of Enterococcus faecalis, Porphyromonas gingivalis and Geobacillus stearothermophilus in BHI were used to contaminate sterile sponges and rotary NiTi instruments (with and without rubber stoppers) inserted into sponges. The various samples were autoclaved and then cultured aerobically and anaerobically. Success of sterilization was measured qualitatively as no growth. The experiment was repeated with clinically used rotary NiTi instruments (n = 512). All experiments were conducted in quadruplicate. RESULTS: No sponges on purchase had microbial growth when anaerobically cultured but some did when aerobically cultured. All autoclaved sponges and instruments (within or without sponges, and with or without rubber stoppers) were associated with no microbial growth. All nonautoclaved positive control samples showed microbial growth. CONCLUSIONS: Autoclaving was effective in the sterilization of sponges and endodontic instruments. Endodontic sponges should be autoclaved before clinical use. For clinical efficiency and cost-effectiveness, rotary NiTi instruments can be sterilized in endodontic sponges without removal of rubber stoppers.

Gingival crevicular fluid proteomes in health, gingivitis and chronic periodontitis.

OBJECTIVE: The aim of this study was to compare the proteome composition of gingival crevicular fluid obtained from healthy periodontium, gingivitis and chronic periodontitis affected sites. BACKGROUND: Owing to its site-specific nature, gingival crevicular fluid is ideal for studying biological processes that occur during periodontal health and disease progression. However, few studies have been conducted into the gingival crevicular fluid proteome due to the small volumes obtained. METHODS: Fifteen males were chosen for each of three different groups, healthy periodontium, gingivitis and chronic periodontitis. They were categorized based on clinical measurements including probing depth, bleeding on probing, plaque index, radiographic bone level, modified gingival index and smoking status. Gingival crevicular fluid was collected from each patient, pooled into healthy, gingivitis and chronic periodontitis groups and their proteome analyzed by gel electrophoresis and liquid chromatography electrospray ionization ion trap tandem mass spectrometry. RESULTS: One hundred and twenty-one proteins in total were identified, and two-thirds of these were identified in all three conditions. Forty-two proteins were considered to have changed in abundance. Of note, cystatin B and cystatin S decreased in abundance from health to gingivitis and further in chronic periodontitis. Complement proteins demonstrated an increase from health to gingivitis followed by a decrease in chronic periodontitis. Immunoglobulins, keratin proteins, fibronectin, lactotransferrin precursor, 14-3-3 protein zeta/delta, neutrophil defensin 3 and alpha-actinin exhibited fluctuations in levels. CONCLUSION: The gingival crevicular fluid proteome in each clinical condition was different and its analysis may assist us in understanding periodontal pathogenesis.

Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 µM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Mass spectrometric analysis of gingival crevicular fluid biomarkers can predict periodontal disease progression.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid has been suggested as a possible source of biomarkers for periodontal disease progression. This paper describes a technique for the analysis of gingival crevicular fluid from individual sites using mass spectrometry. It explores the novel use of mass spectrometry to examine the relationship between the relative amounts of proteins and peptides in gingival crevicular fluid and their relationship with clinical indices and periodontal attachment loss in periodontal maintenance patients. The aim of this paper was to assess whether the mass spectrometric analysis of gingival crevicular fluid may allow for the site-specific prediction of periodontal disease progression. MATERIAL AND METHODS: Forty-one periodontal maintenance subjects were followed over 12 mo, with clinical measurements taken at baseline and every 3 mo thereafter. Gingival crevicular fluid was collected from subjects at each visit and was analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Samples were classified based upon pocket depth, modified gingival index (MGI), plaque index and attachment loss, and were analysed within these groups. A genetic algorithm was used to create a model based on pattern analysis to predict sites undergoing attachment loss. RESULTS: Three hundred and eighty-five gingival crevicular fluid samples were analysed. Twenty-five sites under observation in 14 patients exhibited attachment loss of > 2 mm over the 12-mo period. The clinical indices pocket depth, MGI, plaque levels and bleeding on probing served as poor discriminators of gingival crevicular fluid mass spectra. Models generated from the gingival crevicular fluid mass spectra could predict attachment loss at a site with a high specificity (97% recognition capability and 67% cross-validation). CONCLUSIONS: Gingival crevicular fluid mass spectra could be used to predict sites with attachment loss. The use of algorithm-generated models based on gingival crevicular fluid mass spectra may provide utility in the diagnosis of periodontal disease.

Mineralisation of developmentally hypomineralised human enamel in vitro.

Molar-incisor hypomineralisation (MIH) is a problematic and costly condition. Caries remineralising agents are often recommended for MIH management despite the lack of evidence that these lesions have the capacity for increasing their mineral content. Following surface layer removal ± NaOCl pre-treatment and 14-day exposure to a CPP-ACFP solution at pH 5.5, MIH lesions were analysed using transverse microradiography and polarised light microscopy. Lesions were highly variable but treatment with the remineralising solution increased mineral content (1,828 ± 461 vol% min · µm, %R = 17.7 ± 5.7) and porosity decreased demonstrating the proof of concept that the mineral content of developmentally hypomineralised enamel can be improved after eruption.

A comparative study of carbonate determination in human teeth using Raman spectroscopy.

Carbonate determination in dental apatites such as dentine and enamel is important for studying the dynamics of dental caries and developmental defects of these tissues. Traditionally, these determinations have been performed by acidic digestion with the subsequent measurement of released carbon dioxide gas. As an alternative, Raman spectroscopy has been used for the determination of carbonate in synthetic carbonated apatites with at least four analytical methods published thus far. However, these methods have not been applied to biological apatites. The aim of this comparative study was to test the suitability of these four methods for the determination of B-type carbonate in human enamel and dentine. A method for determining the A-type carbonate content of enamel using the Raman technique is also presented. Raman spectra were obtained from 10 human enamel and dentine samples and analysed with each of the four methods using either a single or multiple ν(1)(PO(4)(3-)) band spectral fitting model. Each of the methods resulted in a different determination for the carbonate content when using the same measurement data. The method that used the full-width-at-half-maximum of the ν(1)(PO(4)(3-)) band to determine the B-type carbonate concentration was found to be in best agreement with (i) the results (using the acid digestion method) of teeth collected from the same sample population and (ii) previously reported values for both enamel and dentine. The use of a multiple-band spectral fitting model produced the highest determination precision (particularly in the case of dentine).

Remineralisation by chewing sugar-free gums in a randomised, controlled in situ trial including dietary intake and gauze to promote plaque formation.

Remineralisation has been shown to be an effective mechanism of preventing the progression of enamel caries. The aim of this double-blind, randomised, cross-over in situ study was to compare enamel remineralisation by chewing sugar-free gum with or without casein phosphopeptide amorphous calcium phosphate (CPP-ACP) where the enamel lesions were exposed to dietary intake and some were covered with gauze to promote plaque formation. Participants wore removable palatal appliances containing 3 recessed enamel half-slabs with subsurface lesions covered with gauze and 3 without gauze. Mineral content was measured by transverse microradiography, and plaque composition was analysed by real-time polymerase chain reaction. For both the gauze-free and gauze-covered lesions, the greatest amount of remineralisation was produced by the CPP-ACP sugar-free gum, followed by the gum without CPP-ACP and then the no-gum control. Recessing the enamel in the appliance allowed plaque accumulation without the need for gauze. There was a trend of less remineralisation and greater variation in mineral content for the gauze-covered lesions. The cell numbers of total bacteria and streptococci were slightly higher in the plaque from the gauze-covered enamel for 2 of the 3 treatment legs; however, there was no significant difference in Streptococcus mutans cell numbers. In conclusion, chewing sugar-free gum containing CPP-ACP promoted greater levels of remineralisation than a sugar-free gum without CPP-ACP or a no-gum control using an in situ remineralisation model including dietary intake irrespective of whether gauze was used to promote plaque formation or not.

Calcium phosphopeptides -- mechanisms of action and evidence for clinical efficacy.

Phosphoproteins/phosphopeptides with clusters of acidic residues are found throughout nature, where they aid in the prevention of unwanted precipitation of solid calcium phosphates. The acidic residues, particularly phosphoserine, interact with calcium and stabilize clusters of calcium and phosphate. Saliva and milk are two examples of biological fluids that contain such phosphoprotein/phosphopeptide-stabilized calcium phosphates, and both share a similar evolutionary pathway. Saliva has been shown to have remineralization potential and is of critical importance in maintaining the mineral content of teeth in the oral environment. Milk can be enzymatically modified to release casein phosphopeptides that contain the clusters of residues that allow milk to stabilize high concentrations of calcium and phosphate. These casein phosphopeptide-stabilized amorphous calcium phosphate nanocomplexes (CPP-ACP) can stabilize even higher concentrations of calcium and phosphate than milk and can be considered a salivary biomimetic, since they share many similarities to statherin. The mechanisms of action and the growing body of scientific evidence that supports the use of CPP-ACP to augment fluoride in inhibiting demineralization and enhancing the remineralization of white-spot lesions are reviewed.

Remineralization models.

During the ICNARA 2 conference, a workshop was held on remineralization models. The group considered the role of remineralization models, whether there was one ideal in situ model design, what essential features should be incorporated into an in situ model, other alternative models, and what new methods for measuring remineralization were on the horizon. This paper summarizes the discussion. In situ and other caries models can be used as a surrogate for caries clinical trials but only when data exist to validate the model. In situ model design should be flexible to allow for investigation of different aspects of the caries process; however, several essential features were identified that should be incorporated into the study design. A range of other caries models was discussed, including the study of non-cavitated lesions, lesions post-orthodontic therapy, plaque retention models to form more standardized lesions, and the study of root caries lesions. Numerous new methods for quantifying remineralization were discussed, but it was considered that these require validation before they can be used in clinical trials.

The Type IX Secretion System and Its Role in Bacterial Function and Pathogenesis.

Porphyromonas, Tannerella, and Prevotella species found in severe periodontitis use the Type IX Secretion System (T9SS) to load their outer membrane surface with an array of virulence factors. These virulence factors are then released on outer membrane vesicles (OMVs), which penetrate the host to dysregulate the immune response to establish a positive feedback loop of chronic, inflammatory destruction of the tooth's supporting tissues. In this review, we present the latest information on the molecular architecture of the T9SS and provide mechanistic insight into its role in secretion and attachment of cargo proteins to produce a virulence coat on cells and OMVs. The recent molecular structures of the T9SS motor comprising PorL and PorM as well as the secretion pore Sov, together with advances in the overall interactome, have provided insight into the possible mechanisms of secretion. We propose the presence of PorL/M motors arranged in a circle at the inner membrane with bent periplasmic rotors interacting with the PorN protein. At the outer membrane, we envisage a slide carousel model where the PorN protein is driven around a circular track composed of PorK. Cargo proteins are transported by PorN to PorW and the Sov translocon just as slides are rotated to the projection window. Secreted proteins are proposed to then be shuttled along highways consisting of the PorV shuttle protein to an array of attachment complexes distributed around the cell. The cell surface attachment of cargo is a hallmark of the T9SS, and in Porphyromonas gingivalis and Tannerella forsythia, this attachment is achieved via covalent bonding to a linking sugar synthesized by the Wbp/Vim pathway. The cell-surface attached cargo are enriched on OMVs, which are then released from the cell.

Microbiome profiles of non-responding and responding paired periodontitis sites within the same participants following non-surgical treatment.

Aim: Periodontitis is a site-specific, chronic disease treated by non-surgical debridement of subgingival plaque. We aimed to determine the microbiome of sites that did not respond to this treatment (NR) compared with paired good responding (GR) sites before and after treatment. Materials and methods: In a longitudinal cohort study, clinical parameters of disease and biological samples were taken prior to and 3 months after treatment. Twelve NR sites from six participants were paired with GR sites within the same participant. Subgingival plaque samples were subjected to bacterial community analysis using 16S rRNA gene sequencing. Results: There were no significant differences in clinical parameters and microbial communities at baseline between GR and NR sites. Bacterial communities in deep pockets were dominated by a small number of species, notably Porphyromonas gingivalis and Treponema denticola. In NR sites three months after treatment there was no significant change in bacterial composition whilst there was a collapse in the abundance of pathobionts in GR sites. Conclusion: NR sites were not identifiable prior to treatment by clinical or microbiological parameters. Treatment failed to disrupt pathogenic bacterial community in NR sites. Targeted suppression of particular species should be considered to initiate community collapse and aid disease resolution.

Casein phosphopeptide-amorphous calcium phosphate incorporated into sugar confections inhibits the progression of enamel subsurface lesions in situ.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been demonstrated to exhibit anticariogenic activity in randomized, controlled clinical trials of sugar-free gum and a tooth cream. Two randomized, double-blind, crossover studies were conducted to investigate the potential of CPP-ACP added to hard candy confections to slow the progression of enamel subsurface lesions in an in situ model. The confections studied were: (1) control sugar (65% sucrose + 33% glucose syrup); (2) control sugar-free; (3) sugar + 0.5% (w/w) CPP-ACP; (4) sugar + 1.0% (w/w) CPP-ACP; (5) sugar-free + 0.5% (w/w) CPP-ACP. Participants (10 and 14 in study 1 and 2) wore a removable palatal appliance containing enamel half-slabs with subsurface lesions, except for meals and oral hygiene procedures, and consumed 1 confection 6 times a day for 10 days. The enamel half-slabs were inset to allow the development of plaque on the enamel surface. Participants rested for 1 week before crossing over to another confection. The appliances were stored in a humid container at 37 degrees C when not in the mouth. After each treatment period, the enamel half-slabs were removed, paired with their demineralized control half-slabs, embedded, sectioned and then analysed using transverse microradiography. In both studies consumption of the control sugar confection resulted in significant demineralization (progression) of the enamel subsurface lesions. However, consumption of the sugar confections containing CPP-ACP did not result in lesion progression, but in fact in significant remineralization (regression) of the lesions. Remineralization by consumption of the sugar + 1.0% CPP-ACP confection was significantly greater than that obtained with the sugar-free confection.

Airway management during in-hospital cardiac arrest: An international, multicentre, retrospective, observational cohort study.

AIM: To determine the type of airway devices used during in-hospital cardiac arrest (IHCA) resuscitation attempts. METHODS: International multicentre retrospective observational study of in-patients aged over 18 years who received chest compressions for cardiac arrest from April 2016 to September 2018. Patients were identified from resuscitation registries and rapid response system databases. Data were collected through review of resuscitation records and hospital notes. Airway devices used during cardiac arrest were recorded as basic (adjuncts or bag-mask), or advanced, including supraglottic airway devices, tracheal tubes or tracheostomies. Descriptive statistics and multivariable regression modelling were used for data analysis. RESULTS: The final analysis included 598 patients. No airway management occurred in 36 (6%), basic airway device use occurred at any time in 562 (94%), basic airway device use without an advanced airway device in 182 (30%), tracheal intubation in 301 (50%), supraglottic airway in 102 (17%), and tracheostomy in 1 (0.2%). There was significant variation in airway device use between centres. The intubation rate ranged between 21% and 90% while supraglottic airway use varied between 1% and 45%. The choice of tracheal intubation vs. supraglottic airway as the second advanced airway device was not associated with immediate survival from the resuscitation attempt (odds ratio 0.81; 95% confidence interval 0.35-1.8). CONCLUSION: There is wide variation in airway device use during resuscitation after IHCA. Only half of patients are intubated before return of spontaneous circulation and many are managed without an advanced airway. Further investigation is needed to determine optimal airway device management strategies during resuscitation following IHCA.

Remineralization and fluoride uptake of white spot lesions under dental varnishes.

INTRODUCTION: The aim of this study was to evaluate white spot lesion (WSL) remineralization and fluoride uptake by the application of fluoride varnishes directly onto artificial WSLs in vitro. METHODS: MI varnish containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and 2.26% fluoride and Duraphat varnish containing 2.26% fluoride (no added calcium) were compared with a placebo varnish (no added calcium or fluoride). Two WSLs were prepared in enamel slabs and varnish applied to cover one of the two lesions. Each slab was immersed in artificial saliva for 14 days at 37°C. Mineral content was determined using transverse microradiography and fluoride uptake using electron probe microanalysis. The data were statistically analysed using a linear mixed model. RESULTS: Both MI and Duraphat varnishes significantly remineralized the covered and uncovered WSLs when compared with the placebo varnish (P < 0.001). The WSLs covered with varnish showed greater remineralization than those uncovered. MI varnish produced the highest level of remineralization and significantly greater fluoride uptake (0.44 ± 0.08 wt%) compared with Duraphat (0.24 ± 0.03 wt%) and the placebo varnish (0.06 ± 0.05 wt%). CONCLUSION: Varnish containing fluoride and CPP-ACP was superior to varnish containing fluoride alone in promoting WSL remineralization and fluoride uptake.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

Progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque.

INTRODUCTION: Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with bacteria. Diagnosis is achieved retrospectively by clinical observation of attachment loss. Predicting disease progression would allow for targeted preventive therapy. The aim of this study was to monitor disease progression in patients on a maintenance program and determine the levels of specific bacteria in subgingival plaque samples and then examine the ability of the clinical parameters of disease and levels of specific bacteria in the plaque samples to predict disease progression. METHODS: During a 12-month longitudinal study of 41 subjects, 25 sites in 21 subjects experienced disease progression indicated by at least 2 mm of clinical attachment loss. Real-time polymerase chain reaction was used to determine the levels of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella intermedia in subgingival plaque samples. RESULTS: No clinical parameters were able to predict periodontal disease progression. In sites undergoing imminent periodontal disease progression within the next 3 months, significant partial correlations were found between P. gingivalis and T. forsythia (r = 0.55, P < 0.001) and T. denticola and T. forsythia (r = 0.43, P = 0.04). The odds of a site undergoing imminent periodontal disease progression increased with increasing levels of P. gingivalis and T. denticola. CONCLUSION: Monitoring the proportions of P. gingivalis and T. denticola in subgingival plaque has the potential to help identify sites at significant risk for progression of periodontitis, which would assist in the targeted treatment of disease.

Anticariogenic efficacy of a saliva biomimetic in head-and-neck cancer patients undergoing radiotherapy.

BACKGROUND: The aim of this study was to investigate the effect of treatment with the saliva biomimetic, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and SnF2 /NaF compared with SnF2 /NaF alone on coronal surface caries progression in head-and-neck cancer patients undergoing radiotherapy. METHODS: Twenty-four participants were randomized into two groups. Both groups used 0.4% SnF2 gel and a 0.32% NaF toothpaste; the test group also applied a crème containing 10% CPP-ACP three times daily while the control group used an identical crème without CPP-ACP (placebo). Resting saliva flow rate and saliva fluoride concentrations were determined. Caries status was assessed using ICDASII at baseline and 12-weeks postradiotherapy. Data were statistically analysed using a linear mixed effects model. RESULTS: Both groups showed significantly reduced resting saliva flow rate (P < 0.001) postradiotherapy. There were no significant differences in flow rates and fluoride concentration between groups. The CPP-ACP group exhibited a significant (P < 0.05) 51% reduction in coronal surface caries progression compared with the placebo group. CONCLUSION: Resting salivary flow rate was significantly reduced in head-and-neck cancer patients following radiotherapy and use of CPP-ACP with SnF2 /NaF significantly lowered caries progression compared with SnF2 /NaF alone.

CPP-ACP Promotes SnF2 Efficacy in a Polymicrobial Caries Model.

Dental caries is associated with plaque dysbiosis, leading to an increase in the proportions of acidogenic and aciduric bacteria at the expense of alkali-generating commensal species. Stannous fluoride (SnF2) slows the progression of caries by remineralization of early lesions but has also been suggested to inhibit glycolysis of aciduric bacteria. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) promotes fluoride remineralization by acting as a salivary biomimetic that releases bioavailable calcium and phosphate ions, and the peptide complex has also been suggested to modify plaque composition. We developed a polymicrobial biofilm model of caries using 6 bacterial species representative of supragingival plaque that were cultured on sound human enamel and pulsed with sucrose 4 times a day to produce a high cariogenic challenge. We used this model to explore the mechanisms of action of SnF2 and CPP-ACP. Bacterial species in the biofilms were enumerated with 16S rRNA gene sequence analyses, and mineral loss and lesion formation were determined in the enamel directly under the polymicrobial biofilms via transverse microradiography. The model tested the twice-daily addition of SnF2, CPP-ACP, or both. SnF2 treatment reduced demineralization by 50% and had a slight effect on the composition of the polymicrobial biofilm. CPP-ACP treatment caused a similar inhibition of enamel demineralization (50%), a decrease in Actinomyces naeslundii and Lactobacillus casei abundance, and an increase in Streptococcus sanguinis and Fusobacterium nucleatum abundance in the polymicrobial biofilm. A combination of SnF2 and CPP-ACP resulted in a greater suppression of the acidogenic and aciduric bacteria and a significant 72% inhibition of enamel demineralization.

Temporal development of the oral microbiome and prediction of early childhood caries.

Human microbiomes are predicted to assemble in a reproducible and ordered manner yet there is limited knowledge on the development of the complex bacterial communities that constitute the oral microbiome. The oral microbiome plays major roles in many oral diseases including early childhood caries (ECC), which afflicts up to 70% of children in some countries. Saliva contains oral bacteria that are indicative of the whole oral microbiome and may have the ability to reflect the dysbiosis in supragingival plaque communities that initiates the clinical manifestations of ECC. The aim of this study was to determine the assembly of the oral microbiome during the first four years of life and compare it with the clinical development of ECC. The oral microbiomes of 134 children enrolled in a birth cohort study were determined at six ages between two months and four years-of-age and their mother's oral microbiome was determined at a single time point. We identified and quantified 356 operational taxonomic units (OTUs) of bacteria in saliva by sequencing the V4 region of the bacterial 16S RNA genes. Bacterial alpha diversity increased from a mean of 31 OTUs in the saliva of infants at 1.9 months-of-age to 84 OTUs at 39 months-of-age. The oral microbiome showed a distinct shift in composition as the children matured. The microbiome data were compared with the clinical development of ECC in the cohort at 39, 48, and 60 months-of-age as determined by ICDAS-II assessment. Streptococcus mutans was the most discriminatory oral bacterial species between health and current disease, with an increased abundance in disease. Overall our study demonstrates an ordered temporal development of the oral microbiome, describes a limited core oral microbiome and indicates that saliva testing of infants may help predict ECC risk.