The effect of dental material type and masticatory forces on periodontitis-derived subgingival microbiomes.

Restorative dental materials can frequently extend below the gingival margin, serving as a potential haven for microbial colonization, and altering the local oral microbiome to ignite infection. However, the contribution of dental materials on driving changes of the composition of the subgingival microbiome is under-investigated. This study evaluated the microbiome-modulating properties of three biomaterials, namely resin dental composites (COM), antimicrobial piezoelectric composites (BTO), and hydroxyapatite (HA), using an optimized in vitro subgingival microbiome model derived from patients with periodontal disease. Dental materials were subjected to static or cyclic loading (mastication forces) during biofilm growth. Microbiome composition was assessed by 16S rRNA gene sequencing. Dysbiosis was measured in terms of subgingival microbial dysbiosis index (SMDI). Biomaterials subjected to cyclic masticatory loads were associated with enhanced biofilm viability except on the antibacterial composite. Biomaterials held static were associated with increased biofilm biomass, especially on HA surfaces. Overall, the microbiome richness (Chao index) was similar for all the biomaterials and loading conditions. However, the microbiome diversity (Shannon index) for the HA beams was significantly different than both composites. In addition, beta diversity analysis revealed significant differences between composites and HA biomaterials, and between both loading conditions (static and cyclic). Under static conditions, microbiomes formed over HA surfaces resulted in increased dysbiosis compared to composites through the enrichment of periopathogens, including Porphyromonas gingivalis, Porphyromonas endodontalis, and Fretibacterium spp., and depletion of commensals such as Granulicatella and Streptococcus spp. Interestingly, cyclic loading reversed the dysbiosis of microbiomes formed over HA (depletion of periopathogenes) but increased the dysbiosis of microbiomes formed over composites (enrichment of Porphyromonas gingivalis and Fusobacterim nucleatum). Comparison of species formed on both composites (control and antibacterial) showed some differences. Commercial composites enriched Selenomonas spp. and depleted Campylobacter concisus. Piezoelectric composites effectively controlled the microbiome viability without significantly impacting the species abundance. Findings of this work open new understandings of the effects of different biomaterials on the modulation of oral biofilms and the relationship with oral subgingival infections.

Bacteriome analysis of Aggregatibacter actinomycetemcomitans-JP2 genotype-associated Grade C periodontitis in Moroccan adolescents.

Background: Grade C (previously aggressive) periodontitis (GCP) in adolescents is prevalent in certain parts of Africa where it is associated with JP2 genotype, a highly virulent strain of Aggregatibacter actinomycetemcomitans. The aim of this study was to characterize the subgingival bacteriome in Moroccan subjects with GCP positive to A. actinomycetemcomitans JP2 genotype. Methods: Subgingival plaque samples were collected from shallow and deep pockets of 8 subjects with GCP (17.2 ± 1.5 years) and from gingival sulci of 13 controls with no periodontitis (14.6 ± 1.1 years). Identification and genotyping of A. actinomycetemcomitans was performed using PCR analysis of the ltx operon, while bacteriome profiling was done by 16S rRNA gene sequencing (V1-V3 region). Groups were compared in terms of microbial diversity, abundances, and dysbiosis. Results: The shallow and deep pocket sites from GCP cases had a significantly altered microbial composition compared to controls. Species associated with health included Haemophilus parainfluenzae, Lautropia mirabilis, Streptococcus spp., Gemella spp., and Rothia spp. While known periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Treponema spp. and Fretibacterium spp., were significantly enriched in GCP, non-conventional taxa, including Pseudomonas oral taxon C61 and Enterobacter cloacae were more abundant and showed stronger association with the disease. Less significant differences in abundances of individual taxa were observed between shallow and deep pockets. Overall dysbiosis measured in terms of Subgingival Microbial Dysbiosis Index (SMDI) differentiated between GCP and no-periodontitis with 95% accuracy. Conclusions: The results suggest that several periodontal pathogens involved in the adult-type periodontitis also play a role in JP2 genotype-associated GCP. The potential role of non-conventional taxa in the pathogenesis of GCP warrants further investigation.

Integrative Metatranscriptomic Analysis Reveals Disease-specific Microbiome-host Interactions in Oral Squamous Cell Carcinoma.

Studies on the microbiome of oral squamous cell carcinoma (OSCC) have been limited to 16S rRNA gene sequencing. Here, laser microdissection coupled with brute-force, deep metatranscriptome sequencing was employed to simultaneously characterize the microbiome and host transcriptomes and predict their interaction in OSCC. The analysis involved 20 HPV16/18-negative OSCC tumor/adjacent normal tissue pairs (TT and ANT) along with deep tongue scrapings from 20 matched healthy controls (HC). Standard bioinformatic tools coupled with in-house algorithms were used to map, analyze, and integrate microbial and host data. Host transcriptome analysis identified enrichment of known cancer-related gene sets, not only in TT versus ANT and HC, but also in the ANT versus HC contrast, consistent with field cancerization. Microbial analysis identified a low abundance yet transcriptionally active, unique multi-kingdom microbiome in OSCC tissues predominated by bacteria and bacteriophages. HC showed a different taxonomic profile yet shared major microbial enzyme classes and pathways with TT/ANT, consistent with functional redundancy. Key taxa enriched in TT/ANT compared with HC were Cutibacterium acnes, Malassezia restricta, Human Herpes Virus 6B, and bacteriophage Yuavirus. Functionally, hyaluronate lyase was overexpressed by C. acnes in TT/ANT. Microbiome-host data integration revealed that OSCC-enriched taxa were associated with upregulation of proliferation-related pathways. In a preliminary in vitro validation experiment, infection of SCC25 oral cancer cells with C. acnes resulted in upregulation of MYC expression. The study provides a new insight into potential mechanisms by which the microbiome can contribute to oral carcinogenesis, which can be validated in future experimental studies. Significance: Studies have shown that a distinct microbiome is associated with OSCC, but how the microbiome functions within the tumor interacts with the host cells remains unclear. By simultaneously characterizing the microbial and host transcriptomes in OSCC and control tissues, the study provides novel insights into microbiome-host interactions in OSCC which can be validated in future mechanistic studies.

Optimization of conditions for in vitro modeling of subgingival normobiosis and dysbiosis.

Modeling subgingival microbiome in health and disease is key to identifying the drivers of dysbiosis and to studying microbiome modulation. Here, we optimize growth conditions of our previously described in vitro subgingival microbiome model. Subgingival plaque samples from healthy and periodontitis subjects were used as inocula to grow normobiotic and dysbiotic microbiomes in MBEC assay plates. Saliva supplemented with 1%, 2%, 3.5%, or 5% (v/v) heat-inactivated human serum was used as a growth medium under shaking or non-shaking conditions. The microbiomes were harvested at 4, 7, 10 or 13 days of growth (384 microbiomes in total) and analyzed by 16S rRNA gene sequencing. Biomass significantly increased as a function of serum concentration and incubation period. Independent of growth conditions, the health- and periodontitis-derived microbiomes clustered separately with their respective inocula. Species richness/diversity slightly increased with time but was adversely affected by higher serum concentrations especially in the periodontitis-derived microbiomes. Microbial dysbiosis increased with time and serum concentration. Porphyromonas and Alloprevotella were substantially enriched in higher serum concentrations at the expense of Streptococcus, Fusobacterium and Prevotella. An increase in Porphyromonas, Bacteroides and Mogibacterium accompanied by a decrease in Prevotella, Catonella, and Gemella were the most prominent changes over time. Shaking had only minor effects. Overall, the health-derived microbiomes grown for 4 days in 1% serum, and periodontitis-derived microbiomes grown for 7 days in 3.5%-5% serum were the most similar to the respective inocula. In conclusion, normobiotic and dysbiostic subgingival microbiomes can be grown reproducibly in saliva supplemented with serum, but time and serum concentration need to be adjusted differently for the health and periodontitis-derived microbiomes to maximize similarity to in vivo inocula. The optimized model could be used to identify drivers of dysbiosis, and to evaluate interventions such as microbiome modulators.

Prolonged mask wearing does not alter the oral microbiome, salivary flow rate or gingival health status - A pilot study.

The COVID-19 pandemic has resulted in the widespread use of N95 respirators and surgical masks, with anecdotal reports among healthcare providers and the public of xerostomia, halitosis, and gingivitis, a consortium of symptoms colloquially termed "mask mouth". However, this has not been scientifically verified. The aim of this study was to assess changes in salivary flow rate, gingival health status and oral microbiome associated with prolonged mask use. A total of 25 dental students (mean age = 26.36 ± 1.58) were included in the study and evaluated at three time points: T1, at the end of at least 2 months of full-day mask wear (7.26 ± 1.56 hours/day); T2, at the end of a period of minimal mask use (1.13 ± 1.13 hours/day); and T3, at the end of 2-3 weeks of resuming full-day mask wear (6.93 ± 1.80 hours/day). Unstimulated whole saliva (UWS) flow rate, xerostomia (on a quantitative scale of 10), gingival index (GI) and plaque index (PI) were assessed at each time point. The salivary microbiome was characterized using 16S rRNA gene sequencing. Overall, UWS flow rates were normal (mean of 0.679 ml/min) and xerostomia, PI and GI scores were low (Mean of 3.11, 0.33 and 0.69, respectively) with no significant differences as a result of prolonged mask wearing. Similarly, there were no significant microbial changes at a false discovery rate (FDR) ≤ 0.05. However, some trends were identified using a nominal p-value cut-off of ≤ 0.01, namely Gemella sanguinis, Streptococcus sp. Oral taxon 066 and Oral taxon 058 were associated with prolonged mask wear. Trends were also seen by gender, race and age, for example an increase in P. gingivalis and P. intermedia with age. In conclusion, we found no evidence that prolonged mask wear adversely affects oral health. The findings support that the oral microbiome of healthy individuals is resilient.

Global transcriptional response of oral squamous cell carcinoma cell lines to health-associated oral bacteria - an in vitro study.

Background: We have recently demonstrated that health-associated oral bacteria Streptococcus mitis, Neisseria flavescens, and Haemophilus parainfluenzae induce cytotoxicity in oral squamous cell carcinoma (OSCC) cell lines and downregulate CD36, a cancer-assocaited gene. Aim: To explore the effect of these three species on global transcriptome of OSCC cell lines. Methods: Gene expression of cell lines CAL27, SCC4 and SCC25 cocultured with the test species was assessed with Clariom-S Human microarray. Porphyromonas gingivalis was included as a pathogenic control. Data were analyzed using Ingenuity Pathway Analysis. Results: The results differed by species and cell line. Overall, the transcriptional changes by S. mitis were predominantly anti-cancer including inhibition of HOTAIR regulatory pathway, JAK/Stat signaling, cyclins/cyclin-dependent kinases, and endothelin1 signaling. H. parainfluenzae and N. flavescens resulted in a mix of pro- and anti-cancer responses including activation of acute phase response, pro-inflammatory interleukins signaling, TREM-1 signaling, and tumor microenvironment pathway; but downregulation of cell cycle by inhibition of cyclins and cyclin-dependent kinases. P. gingivalis had a predominantly pro-cancer effect limited to SCC4, including upregulation of inflammatory pathways, phospholipases and PI3K signaling. Conclusion: These findings provide a new insight into the role of commensal oral bacteria in OSCC. Animal studies are required to further explore them.

Antifungal Effect of Piezoelectric Charges on PMMA Dentures.

Candida-associated denture stomatitis is a recurring disease affecting up to 67% of denture wearers. Poly(methyl methacrylate) (PMMA) remains the main material employed in the fabrication of dentures due to its desirable physical, mechanical, and aesthetic properties. However, the improvement of its antimicrobial properties remains a challenge. To address this need, we developed PMMA composite filled with piezoelectric nanoparticles of barium titanate (BaTiO3) for therapeutic effects. Candida albicans biofilms were cultivated on the surface of the composites under continuous cyclic mechanical loading to activate the piezoelectric charges and to resemble mastication patterns. The interactions between biofilms and biomaterials were evaluated by measuring the biofilm biomass, metabolic activity, and the number of viable cells. To explore the antifungal mechanisms, changes in the expression of genes encoding adhesins and superoxide dismutase were assessed using reverse transcription-polymerase chain reaction. With the addition of piezoelectric nanoparticles, we observed a significant reduction in the biofilm formation and interference in the yeast-to-hyphae transition compared to the standard PMMA. Moreover, we observed that the cyclic deformation of biomaterial surfaces without antifungal agents produced increased biomass, metabolic activity, and a number of viable cells compared to the static/no-deformed surfaces. Cyclic deformation appears to be a novel mechanobiological signal that enables pathogenicity and virulence of C. albicans cells with increased expression of the yeast-to-hyphae transition genes. The outcome of this study opens new opportunities for the design of antifungal dentures for improved clinical service and reduced need for cleaning methods.

Tongue microbiome in children with autism spectrum disorder.

Background: A few recent studies have characterized the salivary microbiome in association with Autism Spectrum Disorder (ASD). Here, we sought to assess if there is an association between the tongue microbiome and ASD. Methods: Tongue scrapping samples were obtained from 25 children with ASD and 38 neurotypical controls. The samples were sequenced for the 16S rRNA gene (V1-V3) and the resultant high-quality reads were assigned to the species-level using our previously described BLASTn-based algorithm. Downstream analyses of microbial profiles were conducted using QIIME, LEfSe, and R. Results: Independent of grouping, Prevotella, Streptococcus, Leptotrichia, Veillonella, Haemophilus and Rothia accounted for > 60% of the average microbiome. Haemophilus parainfluenzae, Rothia mucilaginosa, Prevotella melaninogenica and Neisseria flavescens/subflava were the most abundant species. Species richness and diversity did not significantly differ between the study groups. Thirteen species and three genera were differentially abundant between the two groups, e.g. enrichment of Actinomyces odontolyticus and Actinomyces lingnae and depletion of Campylobacter concisus and Streptococcus vestibularis in the ASD group. However, none of them withstood adjustment for multiple comparisons. Conclusion: The tongue microbiome of children with ASD was not significantly different from that of healthy control children, which is largely consistent with results from the literature.

Salivary Oral Microbiome of Children With Juvenile Idiopathic Arthritis: A Norwegian Cross-Sectional Study.

Background: The oral microbiota has been connected to the pathogenesis of rheumatoid arthritis through activation of mucosal immunity. The objective of this study was to characterize the salivary oral microbiome associated with juvenile idiopathic arthritis (JIA), and correlate it with the disease activity including gingival inflammation. Methods: Fifty-nine patients with JIA (mean age, 12.6 ± 2.7 years) and 34 healthy controls (HC; mean age 12.3 ± 3.0 years) were consecutively recruited in this Norwegian cross-sectional study. Information about demographics, disease activity, medication history, frequency of tooth brushing and a modified version of the gingival bleeding index (GBI) and the simplified oral hygiene index (OHI-S) was obtained. Microbiome profiling of saliva samples was performed by sequencing of the V1-V3 region of the 16S rRNA gene, coupled with a species-level taxonomy assignment algorithm; QIIME, LEfSe and R-package for Spearman correlation matrix were used for downstream analysis. Results: There were no significant differences between JIA and HC in alpha- and beta-diversity. However, differential abundance analysis revealed several taxa to be associated with JIA: TM7-G1, Solobacterium and Mogibacterium at the genus level; and Leptotrichia oral taxon 417, TM7-G1 oral taxon 352 and Capnocytophaga oral taxon 864 among others, at the species level. Haemophilus species, Leptotrichia oral taxon 223, and Bacillus subtilis, were associated with healthy controls. Gemella morbillorum, Leptotrichia sp. oral taxon 498 and Alloprevotella oral taxon 914 correlated positively with the composite juvenile arthritis 10-joint disease activity score (JADAS10), while Campylobacter oral taxon 44 among others, correlated with the number of active joints. Of all microbial markers identified, only Bacillus subtilis and Campylobacter oral taxon 44 maintained false discovery rate (FDR) < 0.1. Conclusions: In this exploratory study of salivary oral microbiome we found similar alpha- and beta-diversity among children with JIA and healthy. Several taxa associated with chronic inflammation were found to be associated with JIA and disease activity, which warrants further investigation.

Screening of Health-Associated Oral Bacteria for Anticancer Properties in vitro.

While extensive literature exists about the role of oral bacterial pathogens like Porphyromonas gingivalis and Fusobacterium nucleatum in oral squamous cell carcinoma (OSCC), the role of health-associated species has been largely unexplored. In this study, we assessed the effect of Streptococcus mitis, Rothia mucilaginosa, Neisseria flavescens, Haemophilus parainfluenzae, Lautropia mirabilis, and Veillonella parvula on proliferation and expression of marker genes (IL-6, TNF-α, MMP3, CD36, CCD1, and NANOG) in OSCC cell lines CAL27, SCC25, and SCC4. Porphyromonas gingivalis was included as a pathogenic control. Both bacterial lysates (3 concentrations) and live cells (3 MOIs) were tested. S. mitis, H. parainfluenzae, and N. flavescens resulted in substantial, dose-dependent reduction of proliferation, which was found to be mediated by H2O2 for the former and intracellular infection in the latter two species. However, only H. parainfluenzae showed differential antiproliferative effect against the cancer cell lines vs. the normal control (TIGKs). In the gene expression assays, the health-associated species mostly downregulated CD36, a gene that plays an important role in tumor growth and metastasis, while P. gingivalis upregulated it. IL6 and TNF expression, on the other hand, was upregulated by almost all species, particularly the Gram-negatives including P. gingivalis. The effect on other genes was less evident and varied significantly by cell line. This exploratory study is the first insight into how health-associated bacteria may interact with OSCC. Further studies to explore whether the observed effects may have implications for the prevention or treatment of oral cancer are warranted.

Tongue microbiome of smokeless tobacco users.

BACKGROUND: The possibility that smokeless tobacco may contribute to oral carcinogenesis by influencing the oral microbiome has not been explored. This preliminary cross-sectional study sought to assess the effect of using shammah, a form of smokeless tobacco prevalent in Arabia, on the tongue microbiome. Tongue scarping samples were obtained from 29 shammah users (SU; 27.34 ± 6.9 years) and 23 shammah non-users (SNU; 27.7 ± 7.19 years) and analyzed with 16S rRNA gene sequencing (V1-V3). Species-level taxonomy assignment of the high-quality, merged reads was obtained using a previously described BLASTn-based algorithm. Downstream analyses were performed with QIIME, LEfSe, and R. RESULTS: A total of 178 species, belonging to 62 genera and 8 phyla were identified. Genera Streptococcus, Leptotrichia, Actinomyces, Veillonella, Haemophilus, Prevotella and Neisseria accounted for more than 60% of the average microbiome. There were no differences between the two groups in species richness and alpha-diversity, but PCoA showed significant separation (P = 0.015, ANOSIM). LEfSe analysis identified 22 species to be differentially abundant between the SU and SNU. However, only 7 species maintained a false discovery rate of ≤0.2 and could cluster the two groups separately: Rothia mucilaginosa, Streptococcus sp. oral taxon 66, Actinomyces meyeri, Streptococcus vestibularis Streptococcus sanguinis and a potentially novel Veillonella species in association with SU, and Oribacterium asaccharolyticum with SNU. CONCLUSION: These preliminary results indicate that shammah use induces tongue microbiome changes including enrichment of several species with high acetaldehyde production potential, which warrants further investigation.

Supragingival mycobiome and inter-kingdom interactions in dental caries.

Background: Recent studies have reveled the presence of a complex fungal community (mycobiome) in the oral cavity. However, the role of oral mycobiome in dental caries and its interaction with caries-associated bacteria is not yet clear. Methods: Whole-mouth supragingival plaque samples from 30 children (6-10 years old) with no caries, early caries, or advanced caries were sequenced for internal transcribed spacer 2 (ITS-2). The mycobiome profiles were correlated with previously published bacteriome counterparts. Interaction among selected fungal and bacterial species was assessed by co-culture or spent media experiments. Results: Fungal load was extremely low. Candida, Malassezia, Cryptococcus, and Trichoderma spp. were the most prevalent/abundant taxa. Advanced caries was associated with significantly higher fungal load and prevalence/abundance of Candida albicans. Cryptococcus neoformans and Candida sake were significantly over-abundant in early caries, while Malassezia globosa was significantly enriched in caries-free subjects. C. albicans correlated with Streptococcus mutans and Scardovia wiggsiae among other caries-associated bacteria, while M. globosa inversely correlated with caries-associated bacteria. In-vitro, M. globosa demonstrated inhibitory properties against S. mutans. Conclusions: the results substantiate the potential role of the oral mycobiome, primarily Candida species, in dental caries. Inter-kingdom correlations and inhibition of S. mutans by M. globosa are worth further investigation.

Subgingival microbiome of experimental gingivitis: shifts associated with the use of chlorhexidine and N-acetyl cysteine mouthwashes.

This study aimed to demonstrate subgingival microbial changes associated with development, prevention, and treatment of experimental gingivitis using chlorhexidine (CHX) and N-acetylcysteine (NAC) mouthwashes. This randomized clinical trial comprised two parts: a 3-week prevention sub-study in which 30 study subjects were equally assigned to either mouthwash or placebo while developing experimental gingivitis; followed by a 2-week treatment sub-study in which 20 subjects with experimental gingivitis were assigned to either mouthwash. Subgingival samples were collected at the beginning and end of each sub-study for microbial profiling with 16S rRNA gene sequencing. As expected, CHX was effective in both preventing and reversing experimental gingivitis; NAC had a modest effect. Gingivitis was associated with enrichment of TM7 HOT-346/349, Tannerella HOT-286, Cardiobacterium valvarum, Campylobacter gracilis, Porphyromonas catoniae, Leptotrichia HOT-219, and Selen o monas spp. At the phylum/genus level, TM7 showed the strongest association. Gingival health was associated with increased abundance of Haemophilus parainfluenzae, Lautropia mirabilis, Rothia spp., Streptococcus spp., and Kingella oralis. CHX demonstrated largely indiscriminate antimicrobial action, resulting in significant drop in biomass and diversity. Our results substantiate the role of specific oral bacterial species in the development of gingivitis. They also indicate that NAC is not a promising mouthwash at the concentration tested.

Metagenome sequencing-based strain-level and functional characterization of supragingival microbiome associated with dental caries in children.

Studies of the microbiome associated with dental caries have largely relied on 16S rRNA sequence analysis, which is associated with PCR biases, low taxonomic resolution, and inability to accurately study functions. Here, we employed whole metagenome shotgun sequencing, coupled with high-resolution analysis algorithm, to analyze supragingival microbiomes from 30 children with or without dental caries. A total of 726 bacterial strains belonging to 406 species, in addition to 34 bacteriophages were identified. A core bacteriome was identified at the species and strain levels. Species of Prevotella, Veillonella, as yet unnamed Actinomyces, and Atopobium showed strongest association with caries; Streptococcus sp. AS14 and Leptotrichia sp. Oral taxon 225, among others, were overabundant in caries-free. For several species, the association was strain-specific. Furthermore, for some species, e.g. Streptococcus mitis and Streptococcus sanguinis, sister strains showed differential associations. Noteworthy, associations were also identified for phages: Streptococcus phage M102 with caries and Haemophilus phage HP1 with caries-free. Functionally, potentially relevant features were identified including urate, vitamin K2, and polyamine biosynthesis in association with caries; and three deiminases and lactate dehydrogenase with health. The results demonstrate new associations between the microbiome and dental caries at the strain and functional levels that need further investigation.

The Impact of the Diurnal Cycle on the Microbial Transcriptome in the Rhizosphere of Barley.

While root exudation follows diurnal rhythms, little is known about the consequences for the microbiome of the rhizosphere. In this study, we used a metatranscriptomic approach to analyze the active microbial communities, before and after sunrise, in the rhizosphere of barley. We detected increased activities of many prokaryotic microbial taxa and functions at the pre-dawn stage, compared to post-dawn. Actinomycetales, Planctomycetales, Rhizobiales, and Burkholderiales were the most abundant and therefore the most active orders in the barley rhizosphere. The latter two, as well as Xanthomonadales, Sphingomonadales, and Caulobacterales showed a significantly higher abundance in pre-dawn samples compared to post-dawn samples. These changes in taxonomy coincide with functional changes as genes involved in both carbohydrate and amino acid metabolism were more abundant in pre-dawn samples compared to post-dawn samples. This study significantly enhances our present knowledge on how rhizospheric microbiota perceives and responds to changes in the soil during dark and light periods.