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.

Longitudinal changes in subgingival biofilm composition following periodontal treatment.

BACKGROUND: Current periodontal treatment involves instrumentation using hand and/or ultrasonic instruments, which are used either alone or in combination based on patient and clinician preference, with comparable clinical outcomes. This study sought to investigate early and later changes in the subgingival biofilm following periodontal treatment, to identify whether these changes were associated with treatment outcomes, and to investigate whether the biofilm responded differently to hand compared with ultrasonic instruments. METHODS: This was a secondary-outcome analysis of a randomized-controlled trial. Thirty-eight periodontitis patients received full-mouth subgingival instrumentation using hand (n = 20) or ultrasonic instrumentation (n = 18). Subgingival plaque was sampled at baseline and 1, 7, and 90 days following treatment. Bacterial DNA was analyzed using 16S rRNA sequencing. Periodontal clinical parameters were evaluated before and after treatment. RESULTS: Biofilm composition was comparable in both (hand and ultrasonics) treatment groups at all time points (all genera and species; p[adjusted] > 0.05). Large-scale changes were observed within groups across time points. At days 1 and 7, taxonomic diversity and dysbiosis were reduced, with an increase in health-associated genera including Streptococcus and Rothia equating to 30% to 40% of the relative abundance. When reassessed at day 90 a subset of samples reformed a microbiome more comparable with baseline, which was independent of instrumentation choice and residual disease. CONCLUSIONS: Hand and ultrasonic instruments induced comparable impacts on the subgingival plaque microbiome. There were marked early changes in the subgingival biofilm composition, although there was limited evidence that community shifts associated with treatment outcomes.

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.

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.

Acetaldehyde production by Rothia mucilaginosa isolates from patients with oral leukoplakia.

Rothia mucilaginosa has been found at high abundance on oral leukoplakia (OLK). The ability of clinical isolates to produce acetaldehyde (ACH) from ethanol has not been investigated. The objective of the current study was to determine the capacity of R. mucilaginosa isolates recovered from OLK to generate ACH. Analysis of R. mucilaginosa genomes (n = 70) shows that this species does not normally encode acetaldehyde dehydrogenase (ALDH) required for detoxification of ACH. The predicted OLK metagenome also exhibited reduced ALDH coding capacity. We analysed ACH production in 8 isolates of R. mucilaginosa and showed that this species is capable of generating ACH in the presence of ethanol. The levels of ACH produced (mean = 53 µM) were comparable to those produced by Neisseria mucosa and Candida albicans in parallel assays. These levels were demonstrated to induce oxidative stress in cultured oral keratinocytes. This study shows that R. mucilaginosa can generate ACH from ethanol in vitro at levels which can induce oxidative stress. This organism likely contributes to oral ACH levels following alcohol consumption and the significance of the increased abundance of R. mucilaginosa in patients with potentially malignant disorders requires further investigation.

Viral infection and oral habits as risk factors for oral squamous cell carcinoma in Yemen: a case-control study.

OBJECTIVE: The role of qat chewing, tobacco (shammah) dipping, smoking, alcohol drinking, and oral viral infection as risk factors for oral squamous cell carcinoma (OSCC) in Yemen was assessed. STUDY DESIGN: A total of 60 cases of OSCC and 120 age- and gender-matched controls were analyzed with respect to demographic data, history of oral habits, and the presence of human papillomavirus (HPV)-16, HPV-18, or Epstein-Barr virus (EBV) as determined by Taqman quantitative polymerase chain reaction. Logistic regression analysis was used to identify independent predictors of the disease. RESULTS: Shammah use was the only risk factor for OSCC, with an odds ratio of 12.6 (CI, 3.3-48.2) and 39 (CI, 14-105) for the ex-users and current users, respectively. The association of shammah use alone with OSCC exceeded that of shammah use in combination with qat chewing, smoking, or both. EBV infection, smoking, and qat chewing showed no association with OSCC, while neither HPV-16 nor HPV-18 were detected in any sample. CONCLUSIONS: Shammah use is a major risk factor for oral cancer in Yemen.

Subgingival periodontal pathogens associated with chronic periodontitis in Yemenis.

BACKGROUND: Subgingival microbial profile associated with periodontitis has been reported to significantly differ by geographical location. The purpose of this study was to assess the association between a panel of putative periodontal bacterial pathogens and chronic periodontitis among Yemenis. METHODS: Subgingival DNA samples were obtained from diseased and healthy sites of 20 non-smoking, moderate to severe chronic periodontitis subjects. Absolute counts (bacterial DNA copies per sample) and relative counts (% total bacteria) of seven periopathogenic species/genera representative of the red and orange complexes were determined using Taqman q-PCR assays. RESULTS: The q-PCR assays showed excellent linearity (R2 > 0.99) and a sensitivity of 100 copies/sample. The detection rate was 100% for all tested species/genera except for P. gingivalis and A. actinomycetemcomitans that were detected at 97.5% and 67.5%, respectively. The median log absolute counts were in the range of 2.41-6.53 copies per sample while median relative counts were in the range of 0.001-0.77%, both being highest for fusobacteria and lowest for A. actinomycetemcomitans. Significant interspecies correlations were observed. Adjusting for multiple comparisons (P≤0.0063), only T. forsythia, T. denticola and P. micra maintained significant association with periodontal destruction. The latter species, however, showed the strongest association and was found in higher proportions at the periodontitis sites across all subjects (3.39 median fold increase). No significant differences were observed for P. gingivalis. CONCLUSIONS: P. micra rather than P. gingivalis appears as a keystone pathogen in this Yemeni Sample. However, these findings need to be validated in a larger-scale study before they can be claimed to represent ethnic variations in pathogens' association with periodontitis.

Normalized real-time PCR for diagnosis of H. pylori infection.

OBJECTIVES: There is increasing interest in the use of quantitative PCR (q-PCR) for diagnosis of H. pylori infection. However, the assay remains largely unstandardized, making comparison between studies unreliable. The objective of this study was to assess accuracy of a normalized q-PCR assay for diagnosis of the infection. SUBJECTS AND METHODS: Seventy-six fresh gastric biopsy specimens were collected from patients undergoing upper gastrointestinal tract endoscopy and examined by rapid urease test (RUT), culture, and a commercial TaqMan q-PCR assay targeting the ureA gene. Counts obtained from the latter assay were normalized to the human ACTB gene. A subject was considered to be infected if two or more assays were positive. RESULTS: The detection rates were 42.1%, 52.6%, and 78.9% by culture, RUT and q-PCR, respectively. Bacterial density ranged 0.005 to 4800 bacteria per 100 human cells. Because q-PCR showed low initial specificity (45.7%), the cutoff value for the assay was recalculated as 1 bacterium per 100 human cells, using ROC curve analysis. Accordingly, the sensitivities and specificities were 79.5% and 97.3%, respectively, for culture; 94.9% and 91.9%, respectively, for RUT; and 94.9% and 94.6%, respectively, for q-PCR. By gold standard, 39 of the dyspeptic patients (51.3%) were found to be infected. CONCLUSIONS: With the identified cutoff value, the q-PCR assay diagnosed H. pylori infection with an accuracy slightly superior to that of RUT. However, the possibility that low counts detected only by q-PCR represent true infections warrants further investigation. Normalization of bacterial counts for standardization of q-PCR H. pylori assays is recommended.

Assessment of Gingival Health Status among 5- and 12-Year-Old Children in Yemen: A Cross-Sectional Study.

Purpose. There are limited data about the gingival health status in Yemeni children. The aim, therefore, was to assess oral hygiene status and prevalence and severity of gingivitis among Yemeni preschool and school children. Materials and Methods. A total of 5396 children were included from 5 representative Yemeni governorates: Sana'a, Hajjah, Hodeida, Hadramaut, and Taiz. Five-year olds (1292) were recruited from private kindergartens while 12-year olds (4104) were selected from public primary schools. Gingival health status was assessed using the plaque index (PI), calculus index (CAI), and gingival index (GI) on the 6 Ramfjord teeth. The latter index was used to categorize gingivitis severity at the subject level. Data were analyzed using simple hypothesis testing, as well as ordinal regression. Results. The 12-year old children had significantly much higher mean PI, CAI, and GI (P < 0.001) with 78.6% presenting with gingivitis and 47.8% with moderate gingivitis. In contrast, the figures were 27.2% and 3.1% in the younger group (P < 0.001). There were significant variations according to gender, area of residence, and governorate. Regression analysis revealed that mean PI (OR = 35), mean CAI (OR = 7.7), male gender (OR = 1.6), living in rural areas (OR = 1.4), and being from Hajjah or Sana'a were independent risk factors of gingivitis severity in the older group. For the 5-year olds, the determinants were mean PI (OR = 122), male gender (OR = 1.4), and living in Sana'a or Taiz. Conclusions. Bad oral hygiene and moderate gingivitis are highly prevalent among Yemeni preschool and school children. Geographical location appeared as important independent risk factors of gingival inflammation.

Khat (Catha edulis)-an updated review.

The habit of chewing fresh leaves and twigs of khat (Catha edulis) for their stimulating amphetamine-like effects is highly prevalent in East Africa and southwest on the Arabic peninsula. There is an extensive literature on khat providing information about its history, botany, production, geographical distribution, chemistry and pharmacology, and exploring the social, economic, medical, psychological and oral aspects related to its use. Some of this literature dates as early as the 11th century; however, most of it appeared after the first scientific description of khat by Peter Forskal in 1775. This review provides a panorama of khat and the various aspects of its use. A non-technical description of the plant chemistry and pharmacology is included. The medical, psychological and oral aspects are emphasized, and the current knowledge about the microbiological effects of khat is also presented.