Host-microbiome interactions in apical periodontitis: The endodontic microbiome in relation to circulatory immunologic markers.

AIM: To explore microbial differences in the endodontic infection of teeth with primary or secondary apical periodontitis (AP), with or without symptomatology. Additionally, to investigate if these differences are depicted in immunologic markers in blood. METHODOLOGY: Twenty-nine teeth with primary or secondary AP were extracted and cryo-pulverized. Blood was drawn from the subjects at three different time-points before and three time-points after the extraction in a time period of four months. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The microbial profiles were ordinated using principal component analysis and tested for differences between groups with permutational multivariate analysis of variance using the Bray-Curtis distance. If significantly different, the microbial profiles were further analysed using the LDA effect size (LEfSe) biomarker discovery tool. A broad panel of inflammatory mediators in blood was examined longitudinally in all subjects during the six visits with mixed models. The Spearman correlation between these mediators and the zOTUs was calculated, and significant correlations (p < .05) were used as input for significant analysis of microarrays (SAM) using MeV. RESULTS: After subsampling, the 467 zOTUs were classified into 9 phyla and 99 genera or higher level taxa. The predominant genus in the entire sample set was Fusobacterium with a relative abundance of 12.3%, followed by Prevotella (9.9%), Actinomyces (7.7%) and Streptococcus (6.7%). The microbiomes of the endodontic infections were significantly associated with endodontic status (primary/secondary infection; p = .015) as well as with the presence or absence of pain (p = .011). There was also a difference in the concentration of inflammatory mediators, namely, C-reactive protein, Interleukin (IL)-8, IL-10, IL-12p70, RANKL and TNF-α, depending on the existence of pain. In addition, the presence of specific bacteria (zOTUs) was correlated, positively or negatively, with the expression of several circulating inflammatory markers. CONCLUSIONS: The microbial profiles and the concentration-time relationship of systemic inflammatory mediators of primary endodontic infection differed from those of secondary, and of symptomatic from those of asymptomatic cases. The fingerprint of associations between the immunological and microbiological profiles differed between asymptomatic and symptomatic patients.

The microbiome of dental and peri-implant subgingival plaque during peri-implant mucositis therapy: A randomized clinical trial.

AIM: To assess the microbial effects of mechanical debridement in conjunction with a mouthrinse on sites with peri-implant mucositis and gingivitis. MATERIALS AND METHODS: Eighty-nine patients with peri-implant mucositis were included in a double-blinded, randomized, placebo-controlled trial with mechanical debridement and 1-month use of either delmopinol, chlorhexidine (CHX), or a placebo mouthrinse. Submucosal and subgingival plaque samples of implants and teeth were collected at baseline and after 1 and 3 months, processed for 16S V4 rRNA gene amplicon sequencing, and analysed bioinformatically. RESULTS: The sites with peri-implant mucositis presented with a less diverse and less anaerobic microbiome. Exposure to delmopinol or CHX, but not to the placebo mouthrinse resulted in microbial changes after 1 month. The healthy sites around the teeth harboured a more diverse and more anaerobe-rich microbiome than the healthy sites around the implants. CONCLUSIONS: Peri-implant sites with mucositis harbour ecologically less complex and less anaerobic biofilms with lower biomass than patient-matched dental sites with gingivitis while eliciting an equal inflammatory response. Adjunctive antimicrobial therapy in addition to mechanical debridement does affect both dental and peri-implant biofilm composition in the short term, resulting in a less dysbiotic subgingival biofilm.

Comparability of microbiota of swabbed and spit saliva.

In general, saliva is used for microbiota analysis in longitudinal studies, and several collection methods are being used. Using a robust sample collection procedure is important, as it may influence salivary composition. This study explored the comparability of the microbiota of swabbed and spit saliva. Twenty-two females participated in this cross-sectional study. The bacterial composition of the three saliva samples (swab collected by the participant (SW-P), swab collected by the researcher (SW-R), and spit (SP) was assessed by 16S rRNA gene amplicon sequencing. The bacterial composition of the swabbed and the spit saliva was significantly different irrespective of the operator, and Shannon diversity was significantly higher in spit saliva than in SW-P and SW-R. The salivary microbiota of spit and swabbed adult saliva differs significantly. Research on microbial composition therefore requires collection of similar saliva sample types in all study participants.

The Effect of Various Fluoride Products on Dentine Lesions during pH-Cycling.

This study compared the effect of topically applied fluoride products on dentine lesions in an in vitro experiment. Demineralized bovine dentine specimens were treated once with either SDF solution (35,400 ppm F), NaF varnish (22,600 ppm F), TiF4 solution (9,200 ppm F), SnF2 gel (1,000 ppm F), no treatment (control), or preserved as baseline lesions. After the application and subsequent removal of the fluoride products, the specimens were subjected to pH-cycling. Calcium loss and uptake in the de- and remineralization buffers were assessed daily. Fluoride release into the buffers was analyzed on days 1, 2, 3, 5, 8, and 13. After the pH-cycling period, mineral distribution throughout the lesion depth was analyzed using transversal microradiography (TMR). X-ray energy-dispersive spectroscopy (EDS) examined the deposition of silver, titanium, and tin after application of SDF, TiF4, and SnF2, respectively. Overall, calcium loss and uptake analysis in the de- and remineralization buffers revealed that the SDF product was the most effective in inhibiting lesion progression, followed by the TiF4, NaF, and SnF2 products. Fluoride analysis disclosed a steep reduction of the amount of fluoride released into de- and remineralization buffers with time. The fluoride effects on de- and remineralization continued beyond the days that fluoride was released into the buffers. TMR analysis showed significant remineralization in the outer zone of the dentine lesions for all fluoride products, with SDF giving hypermineralization in this zone. In the inner zone, lesions developed in all fluoride groups, with the smallest in the SDF group. EDS showed silver and titanium deposition in depth up to 85 µm and 8 µm, respectively, while no tin deposition was observed. The silver in the dentine lesions did not contribute significantly to the density of the TMR profiles in the SDF group. In conclusion, all topical fluoride products protected the dentine lesions against lesion progression, but at different degrees. SDF showed a superior effect in protection against further demineralization and enhancement of remineralization. This was probably attributed to its fluoride concentration that was the highest among the fluoride products.

A review of treatment modalities in gyrate atrophy of the choroid and retina (GACR).

Gyrate atrophy of the choroid and retina (GACR) is a rare inborn error of amino acid metabolism caused by bi-allelic variations in OAT. GACR is characterised by vision decline in early life eventually leading to complete blindness, and high plasma ornithine levels. There is no curative treatment for GACR, although several therapeutic modalities aim to slow progression of the disease by targeting different steps within the ornithine pathway. No international treatment protocol is available. We systematically collected all international literature on therapeutic interventions in GACR to provide an overview of published treatment effects. METHODS: Following the PRISMA guidelines, we conducted a systematic review of the English literature until December 22nd 2020. PubMed and Embase databases were searched for studies related to therapeutic interventions in patients with GACR. RESULTS: A total of 33 studies (n = 107 patients) met the inclusion criteria. Most studies were designed as case reports (n = 27) or case series (n = 4). No randomised controlled trials or large cohort studies were found. Treatments applied were protein-restricted diets, pyridoxine supplementation, creatine or creatine precursor supplementation, l-lysine supplementation, and proline supplementation. Protein-restricted diets lowered ornithine levels ranging from 16.0-91.2%. Pyridoxine responsiveness was reported in 30% of included mutations. Lysine supplementation decreased ornithine levels with 21-34%. Quality assessment showed low to moderate quality of the articles. CONCLUSIONS: Based primarily on case reports ornithine levels can be reduced by using a protein restricted diet, pyridoxine supplementation (variation-dependent) and/or lysine supplementation. The lack of pre-defined clinical outcome measures and structural follow-up in all included studies impeded conclusions on clinical effectiveness. Future research should be aimed at 1) Unravelling the OAT biochemical pathway to identify other possible pathologic metabolites besides ornithine, 2) Pre-defining GACR specific clinical outcome measures, and 3) Establishing an international historical cohort.

Manipulation of Saliva-Derived Microcosm Biofilms To Resemble Dysbiotic Subgingival Microbiota.

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and reestablish a health-associated microbiota, a high-throughput in vitro multispecies biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rRNA gene amplicon sequencing as well as measurement of dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principal-component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis to saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder a microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies.IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed targeting reversing dysbiosis and restoring host-compatible microbiota rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in a dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large-scale evaluation of strategies that can potentially modulate microbial ecology.

Optimizing the quality of clinical studies on oral microbiome: A practical guide for planning, performing, and reporting.

With this review, we aim to increase the quality standards for clinical studies with microbiome as an output parameter. We critically address the existing body of evidence for good quality practices in oral microbiome studies based on 16S rRNA gene amplicon sequencing. First, we discuss the usefulness of microbiome profile analyses. Is a microbiome study actually the best approach for answering the research question? This is followed by addressing the criteria for the most appropriate study design, sample size, and the necessary data (study metadata) that should be collected. Next, we evaluate the available evidence for best practices in sample collection, transport, storage, and DNA isolation. Finally, an overview of possible sequencing options (eg, 16S rRNA gene hypervariable regions, sequencing platforms), processing and data interpretation approaches, as well as requirements for meaningful data storage, sharing, and reporting are provided.

Submucosal microbiome of peri-implant sites: A cross-sectional study.

AIM: To study the peri-implant submucosal microbiome in relation to implant disease status, dentition status, smoking habit, gender, implant location, implant system, time of functional loading, probing pocket depth (PPD), and presence of bleeding on probing. MATERIALS AND METHODS: Biofilm samples were collected from the deepest peri-implant site of 41 patients with paper points, and analysed using 16S rRNA gene pyrosequencing. RESULTS: We observed differences in microbial profiles by PPD, implant disease status, and dentition status. Microbiota in deep pockets included higher proportions of the genera Fusobacterium, Prevotella, and Anaeroglobus compared with shallow pockets that harboured more Rothia, Neisseria, Haemophilus, and Streptococcus. Peri-implantitis (PI) sites were dominated by Fusobacterium and Treponema compared with healthy implants and peri-implant mucositis, which were mostly colonized by Rothia and Streptococcus. Partially edentulous (PE) individuals presented more Fusobacterium, Prevotella, and Rothia, whereas fully edentulous individuals presented more Veillonella and Streptococcus. CONCLUSIONS: PPD, implant disease status, and dentition status may affect the submucosal ecology leading to variation in composition of the microbiome. Deep pockets, PI, and PE individuals were dominated by Gram-negative anaerobic taxa.

The effect of equal fluoride concentrations in silver diamine fluoride and potassium fluoride on demineralized dentin during pH-cycling: chemical data.

The actual contribution of silver in silver diamine fluoride (SDF) towards the anti-demineralizing effect is unclear. This study compared the effects of single applications of three concentrations of fluoride (4.1%, 1.025%, 0.26% F- ) in the form of SDF and potassium fluoride (KF) on demineralized dentin in a 15-day non-microbial pH-cycling model. Calcium loss and uptake in de- and remineralization buffers were analyzed daily. Fluoride release in both buffers was analyzed on days 1, 2, 3, and 8. The net calcium results of de- and remineralization cycles revealed dose-response protection without significant differences between equal fluoride concentrations of SDF and KF. In the demineralization cycles, all fluoride treatments, except KF 0.26% F- , significantly inhibited demineralization, with KF 4.1% F- being the most effective. In the remineralization cycles, remineralization was enhanced in all fluoride concentration groups in a dose-response manner with no difference between similar fluoride concentrations of SDF and KF. Daily fluoride effects were constant throughout the experiment. Fluoride analysis revealed statistically significant differences in fluoride release between the treatments on day 1 that diminished on days 2 and 3. The non-microbial model showed no differences between SDF and KF in inhibiting demineralization and enhancing remineralization of dentin lesions.

Qualitative and quantitative differences in the subgingival microbiome of the restored and unrestored teeth.

BACKGROUND AND OBJECTIVE: Metal-based dental restorations with a subgingival outline may enhance plaque accumulation and bacterial colonization. This study aimed to investigate whether metal-based restorations influence the composition of subgingival microbiome. MATERIAL AND METHODS: Per subject one site with a metal-based restoration and one contra-lateral site without a restoration were selected on basis of radiographic bone loss ≤2 mm, restoration outline at sulcus level/subgingivally, pocket depth ≤4 mm, and no root canal treatments. Subgingival samples were collected with sterile paper-points, and microbial profiles were obtained by 16S rRNA gene amplicon sequencing. Restorations were sampled with an Arkansas-stone and the metal composition was determined using energy-dispersive X-ray spectroscopy. RESULTS: A total of 22 sites from 11 subjects were included. No significant differences for the clinical parameters were found between the restored and unrestored sites. The average age of the restorations was 14.9 ± 7.1 years. Firmicutes was the most prevalent phylum at the restored sites (32% vs 20% of the reads of the unrestored sites, P = 0.016), and Actinobacteria at the unrestored sites (33% vs 18% of the reads of the restored sites, P = 0.01). Overall, sequences clustered into 573 operational taxonomic units (OTUs). Species richness of the restored sites was significantly higher than species richness of the unrestored sites (117 ± 32 and 96 ± 20 OTUs, respectively, P = 0.013). No associations between the metal composition and bacterial profiles were found. CONCLUSION: This study shows that metal-based restorations may enhance colonization of Firmicutes and the neighboring pocket may harbor more diverse microbial communities.

European Organization for Caries Research Workshop: Methodology for Determination of Potentially Available Fluoride in Toothpastes.

Toothpastes are the most universally accepted form of fluoride delivery for caries prevention. To provide anti-caries benefits, they must be able to release fluoride during the time of tooth brushing or post brushing into the oral cavity. However, there is no standard accepted procedure to measure how much fluoride in a toothpaste may be (bio) available for release. The European Organization for Caries Research proposed and supported a workshop with experts in fluoride analysis in toothpastes and representatives from industry. The objective of the workshop was to discuss issues surrounding fluoride analysis in toothpaste and reach consensus on terminology and best practices, wherever the available evidence allowed it. Participants received a background paper and heard presentations followed by structured discussion to define the problem. The group also reviewed evidence on the validity, reliability and feasibility of each technique (namely chromatography and fluoride electroanalysis) and discussed their strengths and limitations. Participants were able to reach a consensus on terminology and were also able to identify and summarize the advantages and disadvantages of each technique. However, they agreed that most currently available methods were developed for regulatory agencies several decades ago, utilizing the best available data from clinical trials then, but require to be updated. They also agreed that although significant advances to our understanding of the mechanism of action of fluoride in toothpaste have been achieved over the past 4 decades, this clearly is an extraordinarily complex subject and more work remains to be done.

Long-term remineralizing effect of MI Paste Plus on regression of early caries after orthodontic fixed appliance treatment: a 12-month follow-up randomized controlled trial.

Background: Casein-phosphopeptide-amorphous-calcium-fluoride-phosphate (CPP-ACFP) can remineralize subsurface lesions. It is the active ingredient of MI-Paste-Plus® (MPP). The long-term remineralization efficacy is unknown. Objective: To evaluate the long-term effect of MPP versus a placebo paste on remineralization of enamel after fixed orthodontic treatment over a 12-month period. Design: This trial was designed as a prospective, double-blinded, placebo-controlled RCT. Methods: Patients with subsurface lesions scheduled for removal of the appliance were included. They applied either MPP or control paste once a day at bedtime for 12 months, complementary to normal oral hygiene. Main outcome measures: Changes in enamel lesions (primary outcome) were fluorescence loss and lesion area determined by quantitative light-induced fluorescence (QLF). Secondary outcomes were Microbial composition, by conventional plating, and acidogenicity of plaque, by capillary ion analysis (CIA), and lesion changes scored visually on clinical photographs. Randomization: Participants [age = 15.5 years (SD = 1.6)] were randomly assigned to either the MPP or the control group, as determined by a computer-randomization scheme, created and locked before the start of the study. Participants received neutral-coloured concealed toothpaste tubes marked A or B. Blinding: The patients and the observers were blinded with respect to the content of tube A or B. Results: A total of 51 patients were analysed; MPP (n = 25) versus control group (n = 26); data loss (n = 14). There was no significant difference between the groups over time for all the used outcome measures. There was a significant improvement in enamel lesions (fluorescence loss) over time in both groups (P < 0.001 and P < 0.001), with no differences between groups. Limitations: Being an in vivo study, non-compliance of the subjects could have influenced the result. Conclusion: The additional use of MPP in patients with subsurface enamel lesions after orthodontic fixed appliance treatment did not improve these lesions during the 1 year following debonding. Registration: This trial is registered at the medical ethical committee of the VU Medical Centre in Amsterdam (NL.199226.029.07).

NGS-eval: NGS Error analysis and novel sequence VAriant detection tooL.

Massively parallel sequencing of microbial genetic markers (MGMs) is used to uncover the species composition in a multitude of ecological niches. These sequencing runs often contain a sample with known composition that can be used to evaluate the sequencing quality or to detect novel sequence variants. With NGS-eval, the reads from such (mock) samples can be used to (i) explore the differences between the reads and their references and to (ii) estimate the sequencing error rate. This tool maps these reads to references and calculates as well as visualizes the different types of sequencing errors. Clearly, sequencing errors can only be accurately calculated if the reference sequences are correct. However, even with known strains, it is not straightforward to select the correct references from databases. We previously analysed a pyrosequencing dataset from a mock sample to estimate sequencing error rates and detected sequence variants in our mock community, allowing us to obtain an accurate error estimation. Here, we demonstrate the variant detection and error analysis capability of NGS-eval with Illumina MiSeq reads from the same mock community. While tailored towards the field of metagenomics, this server can be used for any type of MGM-based reads. NGS-eval is available at http://www.ibi.vu.nl/programs/ngsevalwww/.

Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro.

OBJECTIVE: The aim of this study was to determine the effect of an oxygenating mouthwash compared to two other established mouthwash products on bacterial composition and metabolic activity of oral biofilms in vitro. MATERIAL AND METHODS: Twelve healthy subjects participated as donors. Plaque-saliva mixture inoculated biofilms were grown and treated with 3 different chemotherapeutic mouthwashes [amine fluoride/stannous fluoride (MD), oxygenating agent (AX), chlorhexidine 0.12 % (PA), and water (W)]. Effects of treatments were assessed on biofilm composition (16S rRNA gene amplicon sequencing), production of organic acids (formate, acetate, lactate, propionate, butyrate using capillary electrophoresis), and viability of the remaining biofilm (CFUs). RESULTS: Microbial profiles of biofilms clustered per inoculum donor and were dominated by the genera Veillonella, Streptococcus, and Prevotella. Microbial diversity was only reduced after PA treatment. Significant changes in composition occurred after treatment with AX, resulting in lower proportions of Veillonella and higher proportions of non-mutans streptococci. Production of all organic acids after PA and lactate after MD was significantly lower as compared to W. AX resulted in reduction of acetate, butyrate, and propionate and increase in lactate production (p < 0.05). Viable counts were significantly lower after PA and AX treatments compared to W, while no significant reduction was observed after MD. CONCLUSIONS: All studied mouthwashes affected the in vitro biofilms differently. The effects of the AX treatment were the most prominent which resulted in changes of the bacterial composition and metabolism. CLINICAL IMPLICATIONS: Awareness by the dental team that mouthwashes can change the bacterial composition and metabolism is important when advising its use.

The mycobiome of root canal infections is correlated to the bacteriome.

OBJECTIVES: Bacterial infection of the root canal system causes apical periodontitis. Less is known about the role of fungi in these infections. This study aimed to assess the fungal prevalence, abundance, and diversity of root canal infections, as well as the relation between fungi and bacteria present in different parts of the root canal. MATERIALS AND METHODS: Twenty-six teeth with primary apical periodontitis were extracted, split in apical and coronal root segments, and cryo-pulverized. Bacteriome profiles of 23 teeth were analyzed based on the V3-V4 hypervariable region of the 16S ribosomal RNA gene. Mycobiome profiles of six teeth were analyzed based on the internal transcribed spacer (ITS) 1 or ITS2 region. Samples were sequenced on the Illumina MiSeq platform. RESULTS: A total of 338 bacterial operational taxonomic units (OTUs), 28 ITS1 OTUs, and 24 ITS2 OTUs were identified. Candida and Malassezia were the most frequently identified fungi. No differences could be found between the bacteriome and mycobiome profiles of the apical and coronal root segments. The bacteriome of fungi-positive root segments contained more Actinomyces, Bifidobacterium, four different Lactobacillus OTUs, Propionibacterium, and Streptococcus. A Spearman correlation matrix between bacteriomes and mycobiomes identified no correlations, but separate clusters could be observed. CONCLUSIONS: A considerable proportion of the root canal infections contain fungi, although fungal diversity is limited. However, when fungi are present, the composition of the bacteriome is clearly different. CLINICAL RELEVANCE: Interaction between bacteria and fungi in root canal infections may complicate the infection and require alternative treatment strategies.

The microbiome associated with equine periodontitis and oral health.

Equine periodontal disease is a common and painful condition and its severe form, periodontitis, can lead to tooth loss. Its aetiopathogenesis remains poorly understood despite recent increased awareness of this disorder amongst the veterinary profession. Bacteria have been found to be causative agents of the disease in other species, but current understanding of their role in equine periodontitis is extremely limited. The aim of this study was to use high-throughput sequencing to identify the microbiome associated with equine periodontitis and oral health. Subgingival plaque samples from 24 horses with periodontitis and gingival swabs from 24 orally healthy horses were collected. DNA was extracted from samples, the V3-V4 region of the bacterial 16S rRNA gene amplified by PCR and amplicons sequenced using Illumina MiSeq. Data processing was conducted using USEARCH and QIIME. Diversity analyses were performed with PAST v3.02. Linear discriminant analysis effect size (LEfSe) was used to determine differences between the groups. In total, 1308 OTUs were identified and classified into 356 genera or higher taxa. Microbial profiles at health differed significantly from periodontitis, both in their composition (p < 0.0001, F = 12.24; PERMANOVA) and in microbial diversity (p < 0.001; Mann-Whitney test). Samples from healthy horses were less diverse (1.78, SD 0.74; Shannon diversity index) and were dominated by the genera Gemella and Actinobacillus, while the periodontitis group samples showed higher diversity (3.16, SD 0.98) and were dominated by the genera Prevotella and Veillonella. It is concluded that the microbiomes associated with equine oral health and periodontitis are distinct, with the latter displaying greater microbial diversity.

Nitrate and the Origin of Saliva Influence Composition and Short Chain Fatty Acid Production of Oral Microcosms.

Nitrate is emerging as a possible health benefactor. Especially the microbial conversion of nitrate to nitrite in the oral cavity and the subsequent conversion to nitric oxide in the stomach are of interest in this regard. Yet, how nitrate influences the composition and biochemistry of the oral ecosystem is not fully understood. To investigate the effect of nitrate on oral ecology, we performed a 4-week experiment using the multiplaque artificial mouth (MAM) biofilm model. This model was inoculated with stimulated saliva of two healthy donors. Half of the microcosms (n = 4) received a constant supply of nitrate, while the other half functioned as control (n = 4). Additionally, all microcosms received a nitrate and sucrose pulse, each week, on separate days to measure nitrate reduction and acid formation. The bacterial composition of the microcosms was determined by 16S rDNA sequencing. The origin of the saliva (i.e., donor) showed to be the strongest determinant for the development of the microcosms. The supplementation of nitrate was related to a relatively high abundance of Neisseria in the microcosms of both donors, while Veillonella was highly abundant in the nitrate-supplemented microcosms of only one of the donors. The lactate concentration after sucrose addition was similarly high in all microcosms, irrespective of treatment or donor, while the concentration of butyrate was lower after nitrate addition in the nitrate-receiving microcosms. In conclusion, nitrate influences the composition and biochemistry of oral microcosms, although the result is strongly dependent on the inoculum.

Profiling of Oral and Nasal Microbiome in Children With Cleft Palate.

OBJECTIVE: To identify the oral and nasal microbial profile of cleft palate children and control children and to reveal interrelationships between the microbiome and the high prevalence of infectious diseases. DESIGN: Saliva and nasal samples of 10 cleft palate children and 10 age-matched control children were analyzed. Total microbial genomic DNA was isolated, polymerase chain reaction-denaturing gradient gel electrophoresis was applied to obtain fingerprints, and selected bands on fingerprints were sequenced. RESULTS: The results revealed a significantly lower saliva microbial diversity in cleft children and a different microbial component in both saliva and nares in children with cleft palate. A higher component similarity between the oral and nasal samples was found in the cleft group than in the control group. Lautropia species and Bacillus species were significantly less present among the saliva samples of cleft group. Dolosigranulum species and Bacillus species were significantly fewer in the nasal cavity of cleft group. Streptococcus species became much more predominant in the nasal cavity of the cleft group than in that of the control group. CONCLUSIONS: A disturbed ecological ecosystem is found in oral and nasal microbiome of children with cleft palate as a consequence of the abnormal communication between the two cavities. Further studies are needed to explore the relationship between the disturbed microbiome and diseases.

Determining the Long-term Effect of Antibiotic Administration on the Human Normal Intestinal Microbiota Using Culture and Pyrosequencing Methods.

The purpose of the study was to assess the effect of ciprofloxacin (500 mg twice daily for 10 days) or clindamycin (150 mg 4 times daily for 10 days) on the fecal microbiota of healthy humans for a period of 1 year as compared to placebo. Two different methods, culture and microbiome analysis, were used. Fecal samples were collected for analyses at 6 time-points. The interval needed for the normal microbiota to be normalized after ciprofloxacin or clindamycin treatment differed for various bacterial species. It took 1-12 months to normalize the human microbiota after antibiotic administration, with the most pronounced effect on day 11. Exposure to ciprofloxacin or clindamycin had a strong effect on the diversity of the microbiome, and changes in microbial composition were observed until the 12th month, with the most pronounced microbial shift at month 1. No Clostridium difficile colonization or C. difficile infections were reported. Based on the pyrosequencing results, it appears that clindamycin has more impact than ciprofloxacin on the intestinal microbiota.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.