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.

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.

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.

N-acetyl cysteine versus chlorhexidine mouthwashes in prevention and treatment of experimental gingivitis: a randomized, triple-blind, placebo-controlled clinical trial.

OBJECTIVES: To compare the efficacy of N-acetyl cysteine (NAC) mouthwash with chlorhexidine (CHX) in prevention and treatment of experimental gingivitis MATERIALS AND METHODS: Sixty subjects were assigned randomly and blindly into one of three equal groups: NAC, CHX, or placebo group. The study was conducted in two stages: preventive and treatment substudies. Professional prophylaxis was performed ahead of starting the preventive substudy. Then, the subjects were instructed to stop oral hygiene practices and begin rinsing twice/day with 15 ml of the assigned mouthwash (1.25% NAC, 0.2% CHX, or inert base). Plaque index (PI), gingival index (GI), and papillary bleeding index (PBI) were measured at baseline, 7, 14, and 21 days. The treatment substudy started on day 21 in which the subjects in the placebo group (now with established experimental gingivitis) were assigned to NAC (n = 10) or CHX (n = 10); the abovementioned indices were measured at 28 and 35 days. Efficacy of these interventions was compared. RESULTS: All groups accumulated plaque and developed some degree of gingivitis: full-blown in the placebo group and remarkably mild in the CHX group. NAC had slight preventive properties at days 14 and 21. In the treatment substudy, CHX was associated with remarkable reduction in plaque and gingivitis while NAC resulted in insignificant reductions. CONCLUSIONS: 1.25% NAC is marginally effective in prevention and treatment of experimental gingivitis. CLINICAL RELEVANCE: When compared with the placebo, NAC showed promising preventive and treatment effects of gingivitis that deserve further development and studies. TRIAL REGISTRATION: ISRCTN31352091.

A dysbiotic mycobiome dominated by Candida albicans is identified within oral squamous-cell carcinomas.

The aim of this study was to characterize the mycobiome associated with oral squamous-cell carcinoma (OSCC). DNA was extracted from 52 tissue biopsies (cases: 25 OSCC; controls: 27 intra-oral fibro-epithelial polyps [FEP]) and sequenced for the fungal internal transcribed spacer 2 region using Illumina™ 2 x300bp chemistry. Merged reads were classified to species level using a BLASTN-algorithm with UNITE's named species sequences as reference. Downstream analyses were performed using QIIME™ and linear discriminant analysis effect size. A total of 364 species representing 160 genera and two phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only five species and four genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. Genera Candida, Hannaella, and Gibberella were overrepresented in OSCC; Alternaria and  Trametes  were more abundant in FEP. Species-wise, Candida albicans, Candida etchellsii, and a Hannaella luteola-like species were enriched in OSCC, while aHanseniaspora uvarum-like species, Malassezia restricta, and Aspergillus tamarii were the most significantly abundant in FEP. In conclusion, a dysbiotic mycobiome dominated by C. albicans was found in association with OSCC, a finding worth further investigation.

Inflammatory bacteriome featuring Fusobacterium nucleatum and Pseudomonas aeruginosa identified in association with oral squamous cell carcinoma.

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional potential of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) was sequenced for the V1-V3 region using Illumina's 2 × 300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp. Oral taxon 44, while Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functional prediction showed that genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F. nucleatum and P. aeruginosa with OSCC. Functionally, an "inflammatory bacteriome" is enriched in OSSC.

Differences in the Bacteriome of Smokeless Tobacco Products with Different Oral Carcinogenicity: Compositional and Predicted Functional Analysis.

Smokeless tobacco (ST) products vary significantly in their oral carcinogenicity. Much is known about the differences in the chemical, but not the bacterial, constituents of these products. In this study, we explored the composition and function of the bacteriome in ST products from four countries using quantitative polymerase chain reaction (qPCR) and 16S rRNA-based next generation sequencing. The bacterial load (16S rRNA copies/gram) was lowest in Swedish snus (3.4 × 106) and highest in Yemeni shammah (6.6 × 1011). A total of 491 species-level taxa, many of which are potentially novel, belonging to 178 genera and 11 phyla were identified. Species richness and diversity were highest for Swedish snus and lowest for Yemeni shammah. Bacillus, Paenibacillus, and Oceanobacillus spp. were the most abundant in American snuff; species of Pseudomonas, Massilia, Propionibacterium, Puniceispirillum, and Gloeothece predominated in Swedish snus. In Sudanese toombak, Facklamia, Desemzia, Atopostipes, and Lysinibacillus spp. accounted for the majority of the bacteriome. Yemeni shammah exclusively contained Bacillus spp. Functional prediction by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that genes encoding cadmium/zinc and nickel transport systems were enriched in the presumptively "high carcinogenicity" products. The bacteriome of ST products thus differed qualitatively, quantitatively, and functionally. The relevance of these differences, particularly with respect to nickel and cadmium, to oral carcinogenesis warrants further investigation.

Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria.

Oral cancer, primarily oral squamous cell carcinoma (OSCC), continues to be a major global health problem with high incidence and low survival rates. While the major risk factors for this malignancy, mostly lifestyle related, have been identified, around 15% of oral cancer cases remain unexplained. In light of evidence implicating bacteria in the aetiology of some cancer types, several epidemiological studies have been conducted in the last decade, employing methodologies ranging from traditional culture techniques to 16S rRNA metagenomics, to assess the possible role of bacteria in OSCC. While these studies have demonstrated differences in microbial composition between cancerous and healthy tissues, they have failed to agree on specific bacteria or patterns of oral microbial dysbiosis to implicate in OSCC. On the contrary, some oral taxa, particularly Porphyromonas gingivalis and Fusobacterium nucleatum, show strong oral carcinogenic potential in vitro and in animal studies. Bacteria are thought to contribute to oral carcinogenesis via inhibition of apoptosis, activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation, and production of carcinogens. This narrative review provides a critical analysis of and an update on the association between bacteria and oral carcinogenesis and the possible mechanisms underlying it.

Antibiotic Prescription Knowledge of Dentists in Kingdom of Saudi Arabia: An Online, Country-wide Survey.

AIM: Dentists are probably contributing to the development of bacterial resistance to certain antibiotics. Campaigns to promote prudent use of antibiotics in dentistry are, thus, needed but require proper identification of dentists' knowledge gaps. The objective here was to comprehensively evaluate antibiotic prescription knowledge of dentists in Saudi Arabia. MATERIAL AND METHODS: A link to an online, previously validated questionnaire was emailed to 5199 dentists registered with the Saudi Dental Society. The questionnaire comprised 42 scorable items measuring antibiotics prescription knowledge in five different domains in addition to nonscorable questions regarding first-choice antibiotics and previous attendance of a course/workshop about antibiotic prescription. Each correct answer was given one mark. Mean scores were calculated as percentages and categorized as good (> 80%), intermediate (60-80%), or poor (< 60%). RESULTS: The response rate was 9.4%; however, only 373 (7.2%) fully completed the questionnaire. Around half of the participants (52%) reported prescribing amoxicillin/clavulanate as the first-choice antibiotic; 62% reported attending a course/workshop in the last 5 years. The average knowledge score was 69%, being highest for nonclinical indications (79%) and lowest for prophylactic use (56%). The worst per-item scores were noted for rheumatic heart disease (19%), trismus (28%), surgical extraction (30%), apicectomy (31%), and periodontal abscess (33%). Female dentists, dentists in governmental sector, and those with higher qualifications had significantly better knowledge. CONCLUSION: The level of knowledge was hardly intermediate and several deficits were identified, indicating an urgent need for educational campaigns and provision of guidelines promoting rational use of antibiotics by dentists. CLINICAL SIGNIFICANCE: Irrational use of antibiotics by dentists can contribute to the problem of antibacterial resistance.

Species-level core oral bacteriome identified by 16S rRNA pyrosequencing in a healthy young Arab population.

BACKGROUND: Reports on the composition of oral bacteriome in Arabs are lacking. In addition, the majority of previous studies on other ethnic groups have been limited by low-resolution taxonomic assignment of next-generation sequencing reads. Furthermore, there has been a conflict about the existence of a 'core' bacteriome. OBJECTIVE: The objective of this study was to characterize the healthy core oral bacteriome in a young Arab population at the species level. METHODS: Oral rinse DNA samples obtained from 12 stringently selected healthy young subjects of Arab origin were pyrosequenced (454's FLX chemistry) for the bacterial 16S V1-V3 hypervariable region at an average depth of 11,500 reads. High-quality, non-chimeric reads ≥380 bp were classified to the species level using the recently described, prioritized, multistage assignment algorithm. A core bacteriome was defined as taxa present in at least 11 samples. The Chao2, abundance-based coverage estimator (ACE), and Shannon indices were computed to assess species richness and diversity. RESULTS: Overall, 557 species-level taxa (211±42 per subject) were identified, representing 122 genera and 13 phyla. The core bacteriome comprised 55 species-level taxa belonging to 30 genera and 7 phyla, namely Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Fusobacteria, Saccharibacteria, and SR1. The core species constituted between 67 and 87% of the individual bacteriomes. However, the abundances differed by up to three orders of magnitude among the study subjects. On average, Streptococcus mitis, Rothia mucilaginosa, Haemophilus parainfluenzae, Neisseria flavescence/subflava group, Prevotella melaninogenica, and Veillonella parvula group were the most abundant. Streptococcus sp. C300, a taxon never reported in the oral cavity, was identified as a core species. Species richness was estimated at 586 (Chao2) and 614 (ACE) species, whereas diversity (Shannon index) averaged at 3.99. CONCLUSIONS: A species-level core oral bacteriome representing the majority of reads was identified, which can serve as a reference for comparison with oral bacteriomes of other populations as well as those associated with disease.

Exome sequencing of oral squamous cell carcinoma in users of Arabian snuff reveals novel candidates for driver genes.

The study sought to identify genetic aberrations driving oral squamous cell carcinoma (OSCC) development among users of shammah, an Arabian preparation of smokeless tobacco. Twenty archival OSCC samples, 15 of which with a history of shammah exposure, were whole-exome sequenced at an average depth of 127×. Somatic mutations were identified using a novel, matched controls-independent filtration algorithm. CODEX and Exomedepth coupled with a novel, Database of Genomic Variant-based filter were employed to call somatic gene-copy number variations. Significantly mutated genes were identified with Oncodrive FM and the Youn and Simon's method. Candidate driver genes were nominated based on Gene Set Enrichment Analysis. The observed mutational spectrum was similar to that reported by the TCGA project. In addition to confirming known genes of OSCC (TP53, CDKNA2, CASP8, PIK3CA, HRAS, FAT1, TP63, CCND1 and FADD) the analysis identified several candidate novel driver events including mutations of NOTCH3, CSMD3, CRB1, CLTCL1, OSMR and TRPM2, amplification of the proto-oncogenes FOSL1, RELA, TRAF6, MDM2, FRS2 and BAG1, and deletion of the recently described tumor suppressor SMARCC1. Analysis also revealed significantly altered pathways not previously implicated in OSCC including Oncostatin-M signalling pathway, AP-1 and C-MYB transcription networks and endocytosis. There was a trend for higher number of mutations, amplifications and driver events in samples with history of shammah exposure particularly those that tested EBV positive, suggesting an interaction between tobacco exposure and EBV. The work provides further evidence for the genetic heterogeneity of oral cancer and suggests shammah-associated OSCC is characterized by extensive amplification of oncogenes.

Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples.

BACKGROUND: Usefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify reads to the species level. OBJECTIVE: The purpose of this study was to develop a robust algorithm for species-level classification of NGS reads from oral samples and to pilot test it for profiling bacteria within OSCC tissues. METHODS: Bacterial 16S V1-V3 libraries were prepared from three OSCC DNA samples and sequenced using 454's FLX chemistry. High-quality, well-aligned, and non-chimeric reads ≥350 bp were classified using a novel, multi-stage algorithm that involves matching reads to reference sequences in revised versions of the Human Oral Microbiome Database (HOMD), HOMD extended (HOMDEXT), and Greengene Gold (GGG) at alignment coverage and percentage identity ≥98%, followed by assignment to species level based on top hit reference sequences. Priority was given to hits in HOMD, then HOMDEXT and finally GGG. Unmatched reads were subject to operational taxonomic unit analysis. RESULTS: Nearly, 92.8% of the reads were matched to updated-HOMD 13.2, 1.83% to trusted-HOMDEXT, and 1.36% to modified-GGG. Of all matched reads, 99.6% were classified to species level. A total of 228 species-level taxa were identified, representing 11 phyla; the most abundant were Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. Thirty-five species-level taxa were detected in all samples. On average, Prevotella oris, Neisseria flava, Neisseria flavescens/subflava, Fusobacterium nucleatum ss polymorphum, Aggregatibacter segnis, Streptococcus mitis, and Fusobacterium periodontium were the most abundant. Bacteroides fragilis, a species rarely isolated from the oral cavity, was detected in two samples. CONCLUSION: This multi-stage algorithm maximizes the fraction of reads classified to the species level while ensuring reliable classification by giving priority to the human, oral reference set. Applying the algorithm to OSCC samples revealed high diversity. In addition to oral taxa, a number of human, non-oral taxa were also identified, some of which are rarely detected in the oral cavity.

Qat Chewing and Periodontal Pathogens in Health and Disease: Further Evidence for a Prebiotic-Like Effect.

AIM: Qat chewing has been reported to induce subgingival microbial shifts suggestive of prebiotic-like properties. The objective here was to assess the effect of qat chewing on a panel of classical and new putative periopathogens in health and periodontitis. MATERIALS AND METHODS: 40 qat chewers and 40 nonchewers, equally stratified by periodontal health status, were recruited. Taqman, real-time PCR was used to quantify total bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Parvimonas micra, Filifactor alocis, Synergistetes, and TM7s in pooled subgingival biofilm samples. Differences in microbial parameters between the study groups were analysed using ordinal regression. RESULTS: In health, the qat chewers harboured significantly lower relative counts of P. gingivalis, T. forsythia, Synergistetes, and TM7s after adjustment for multiple comparisons (P ≤ 0.007). At nominal significance level, they also carried lower counts of TM7s and P. micra (P ≤ 0.05). In periodontitis, the chewers had lower counts of all taxa; however, only T. denticola withstood correction for multiple comparisons (P ≤ 0.0063). CONCLUSIONS: Qat chewing is associated with lower proportions of periopathogens, particularly in subjects with healthy periodontium, which supports previous reports of its prebiotic-like properties. This potentially beneficial biological effect can be exploited by attempting to isolate the active fraction.

Salivary Candida species carriage patterns and their relation to caries experience among yemeni children.

PURPOSE: This study was carried out to assess the carriage rates, counts and species distribution of Candida in saliva of 6- to 12-year-old Yemeni children and relate that to their caries experience. MATERIALS AND METHODS: One hundred eighty children were recruited. Oral hygiene and caries were assessed using the simplified oral hygiene index and dft/DMFT index, respectively. Detection and quantification of 4 Candida species in unstimulated saliva were performed using CHROMagar Candida medium. Data were analysed using regression analysis. RESULTS: Candida was detected in 60% of the children with a mean count of 923 ± 1875 CFU/ml. C. albicans accounted for 60% of the isolates and was the only species to be detected with more than 1000 CFU/ml. Non-albicans candida and unidentified species represented 16.3% and 23.1% of the isolates, respectively. One novel finding was that a significant proportion (38%) of the carriers harboured two or more species, which for the first time allowed the identification of four age-dependent carriage patterns (clusters). Another somewhat new observation was that carriage at ≥ 1000 CFU/ml in particular significantly correlated with caries in primary and permanent dentitions (r = 0.23 and 0.18, respectively) as well as a caries-active status (OR = 6.9). Interestingly, the C. glabrata cluster had significantly lower primary caries scores than other clusters. CONCLUSIONS: The findings substantiate claims of geographical variations in candida carriage and the relation between candida carriage and caries. The validity of carrier clusters and the use of 1000 CFU/ml as a risk marker should be further investigated.