Label-Free Quantitative Proteomics of Oral Microbial Communities.

The oral cavity is a habitat for different microorganisms, of which bacteria are best described. Studying different bacterial taxa and their proteins is crucial to understanding their interactions with the host and other microbes. Also, for bacteria with virulence potential, identifying novel antigenic proteins is essential to finding candidates for the development of vaccines.Here, a workflow for gel-free and label-free protein analysis of oral bacterial species grown in vitro as a biofilm and a planktonic culture is described. Details on cultivation, protein extraction and digestion, peptide cleanup, LC-MS/MS run parameters, and subsequent bioinformatics analysis are included. Challenging steps in the workflow, such as growing different types of bacteria and selecting a suitable protein database, are also discussed. This protocol provides a valuable guide for metaproteomic experiments using multi-species models of oral bacteria.

The Prevalence of Smokeless Tobacco Consumption and Its Effects on Gingival and Periodontal Health Among the Adults in the Emirates of Sharjah, UAE - A Cross-Sectional Study.

Introduction: Smokeless tobacco (SLT) has been linked with various oral signs and symptoms, such as oral mucosal lesions and gingival and periodontal effects. However, there is limited research on the prevalence of SLT use and its impact on gingival and periodontal health in the adult population of the Emirates of Sharjah. Aim: To assess the prevalence of SLT consumption and its effects on gingival and periodontal health in the adult population of the Emirates of Sharjah. Methods: The study evaluated 171 dentate patients aged 18-77 years who use SLT or are nonsmokers. Data were collected through a questionnaire on SLT use and periodontal health assessed using plaque index, gingival index (GI), attachment score, and gingival recession (GR). Statistical analysis was done using SPSS 27.0 version. Results: Among 390 screened participants, 28 subjects (21 males and 7 females) were SLT users with prevalence of 7.18%. No significant differences were found in plaque index, GI, or periodontal pocket depth. However, SLT users had significantly higher clinical attachment loss scores (mean 4.01 mm, SD 0.35) compared to non-users (mean 1.49 mm, SD 1.12). Significant differences were also observed in GR. Conclusions: This study provides insights into the prevalence and impact of SLT use on periodontal and gingival health in UAE adults. SLT usage is associated with a higher risk of periodontitis and gingivitis, indicating the need for awareness and intervention. Further research is required to validate results and consider confounding factors.

The impact of periodontal disease on the clinical outcomes of COVID-19: A systematic review and meta-analysis.

BACKGROUND: A possible relationship between periodontitis (PD) and COVID-19 and its adverse outcomes has been suggested. Hence, the present systematic review and meta-analysis aimed to investigate the available evidence regarding the potential association between periodontitis (PD) and COVID-19 and its adverse outcomes. MATERIALS AND METHODS: PubMed, Scopus, Web of Science, and Google Scholar were searched for relevant studies published up to April 15th, 2023. Studies that evaluated the association between PD and COVID-19 were included. Risk of bias was evaluated by two reviewers, and meta-analyses were performed using RevMan 5.3 software. RESULTS: A total of 22 studies involving 92,535 patients from USA, Europe, Asia, the Middle East and South America were included; of these, 12 were pooled into the meta-analysis. Most of the studies (19 studies) reported a significant association between PD and COVID-19. The pooled data found a significant association between PD and COVID-19 outcomes: more severe symptoms (OR = 6.95, P = 0.0008), ICU admissions (OR = 3.15, P = 0.0001), and mortality (OR = 1.92, P = 0.21). Additionally, compared to mild PD, severe PD was significantly associated with higher risks of severe COVID-19 outcomes: severe symptoms (P = 0.02); ICU admission (P = 0.0001); and higher mortality rates (P = 0.0001). The results also revealed 58% higher risk for COVID-19 infection in patients with PD (P = 0.00001). CONCLUSIONS: The present findings suggest a possible association between poor periodontal health and the risk of poor COVID-19 outcomes. However, owing to the observed methodological heterogeneity across the included studies, further prospective cohort studies with standardized methodologies are warranted to further unravel the potential association between periodontal disease and COVID-19 and its adverse outcomes.

Oral cancer knowledge, attitudes, and practices among senior dental students in Yemen: a multi-institution study.

BACKGROUND: The "Knowledge, attitude, and practice (KAP)" approach is crucial in health system. Appreciating the current KAP status will reveal the extent of the efficiency of applied health strategies, and subsequently help to determine the appropriate health policy to be employed for improving the health indicators of a given disease/condition, including Oral Cancer (OC). This large-scale cross-sectional study aimed to assess knowledge, attitude, and practice on OC among senior dental students in Yemen. METHODS: A pre-validated online questionnaire was used for data collection. It consisted of a series of close-ended questions on knowledge, attitudes and practices related to OC. Yemeni dental students in clinical levels (4th and 5th years) from nine dental schools based in four major cities were invited to complete the survey. The SPSS Version 28.0 was used for data analysis. Differences by different grouping factors were assessed by Chi-squared and Mann Whitney-U tests, as appropriate. RESULTS: A total of 927 students completed the questionnaire: a response rate of 43%. While the majority identified smoking (93.8%), and smokeless tobacco (92.1%) as potential risk factors of OC, only 76.2% recognized sun exposure as a risk factor for lip cancer and only 50% were aware of old age as a risk factor of OC. Regarding clinical signs of OC, 84.1% reported that OC can present as non-healing ulcer, but only two thirds of the participants recognized that OC can present as a white and/or red lesion. With respect to practices, although 92.1% reported asking their patients regarding oral habits, only 78% reported they regularly carry out a soft tissue examination. Only 54.5% of the participants considered themselves to be well-trained to provide smoking cessation advice, and 21% were confident regarding their knowledge on OC. The 5th year students showed significantly better knowledge and practices than the 4th year students did (p < 0.01). CONCLUSION: The study suggests significant gaps in knowledge, attitudes, and practices of senior dental students in Yemen regarding OC. The findings also underscore the urgent need to improve OC teaching and training of undergraduate dental students, and to provide periodic well-structured continuing professional development activities for dental professionals.

The Association Between Periodontal Disease and Chronic Migraine: A Systematic Review.

Migraine is a neurologic illness that produces intense throbbing pain on one side of the head and affects roughly 1 billion people worldwide. Recent research indicates a relationship between periodontitis and chronic migraines. This study aimed to review the association between chronic migraines and periodontitis through a systematic literature review. Four research databases (Google Scholar, PubMed, ProQuest, and SpringerLink) were searched according to PRISMA guidelines to retrieve the studies included in this review. A search strategy was developed to answer the study question with appropriate inclusion and exclusion criteria. Out of 34 published studies, 8 studies were included in this review. Three of the studies were cross-sectional, 3 were case-control, and 2 were clinical report and medical hypothesis papers. Seven of the 8 included studies showed that there is an association between periodontal disease and chronic migraine. The elevated blood levels of some biomarkers such as leptins, ProCalcitonin (proCT), calcitonin gene-related peptides (CGRPs), Pentraxin 3 (PTX3), and Soluble Tumor Necrosis Factor-like Weak Inducer Of Apoptosis (sTWEAK) play a significant role in this association. The limitations include a small sample size, the influence of anti-inflammatory drugs, and a self-reported headache measure that is subject to misclassification bias. This systematic review reveals a supposed correlation between periodontal disease and chronic migraine, as evidenced by various biomarkers and inflammatory mediators. This suggests that periodontal disease could potentially contribute to the development of chronic migraine. However, to further assess the potential benefits of periodontal treatment in patients with chronic migraine, additional longitudinal studies with larger sample sizes and interventional studies are needed.

The feasibility of needleless jet injection versus conventional needle local anesthesia during dental procedures: a systematic review.

This systematic review evaluates current evidence regarding the feasibility of using needleless jet injection instead of a conventional local anesthetic needle. EBSCO, ProQuest, PubMed, and Scopus databases were used to identify relevant literature published in English from 2005 to 2020. Ten studies were selected. Five of them were randomized clinical trials, 3 case-control studies, and 2 equivalence trials. Using the Critical Appraisal Skills Program checklist, 6 studies scored between 67% and 100%, and 4 studies scored between 34% and 66%. According to Jadad's scale, 2 studies were considered strong, and 8 studies were considered moderate in quality. The results of the 10 studies showed differences in patient preference for needleless jet injection. Needleless injection technique has been found to be particularly useful in uncooperative patients with anxiety and needle phobia. Needleless jet injection is not technique sensitive. However, with needleless jet anesthesia, most treatments require additional anesthesia. Conventional needle anesthesia is less costly, has a longer duration of action, and has better pain control during dental extraction. Needleless jet anesthesia has been shown to be moderately accepted by patients with a fear of needles, has a faster onset of action, and is an efficient alternative to conventional infiltration anesthesia technique.

Khat (Catha Edulis Forsk) induced apoptosis and cytotoxicity in cultured cells: A scoping review.

BACKGROUND: Khat (Catha edulis Forsk) leaves are chewed by people in certain regions of East Africa and the Middle East for their stimulating amphetamine-like effects. The purpose of this scoping review is to systematically map the current in vitro publications that investigated the toxicological potential effects of khat on cultured human or animal cells in terms of cellular viability and activity. METHODS: A comprehensive electronic database search was undertaken up to December 2020 without starting date or language restrictions in accordance with the PRISMA extension for scoping review guideline and methodological quality evaluation based on the guidelines for reporting pre-clinical in vitro studies on dental materials. All in vitro studies that investigated the effect of khat plant extract (Catha Edulis) on the cultured human or animal cells were included. RESULTS: The initial search yielded 599 articles and 16 articles were finally selected to be included. The treatment of cells with khat produced different degrees of cellular changes, including decreased cellular survival, induction of apoptosis, increased ROS production, alteration of cell phenotype, and of arrest cell cycle. In this contest, khat-exposed cells expressed higher levels of pro-apoptotic protein Bax and lower levels of anti-apoptotic Bcl-2, up-regulated p38, p53, p16, and p21 proteins, as well as premature expression of differentiation markers. CONCLUSION: Based on the current scoping review, khat induced apoptosis and cytotoxicity in cultured human cells, including oral cells.

Tongue-coating microbiome as a cancer predictor: A scoping review.

OBJECTIVE: The tongue microbiome has emerged as a non-invasive diagnostic and tracking prognostic tool in the detection of diseases mainly cancer. This scoping review aimed to identify the association between tongue microbiome and pre-cancer or cancer lesions. DESIGN: A comprehensive electronic database search including PubMed, Web of Science, and Scopus was undertaken up to March 2021, without language or date restrictions. This review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guideline. All observational studies that compared microbial community on the dorsal surface of the tongue between cancer or precancerous cases and healthy controls using NGS techniques were included. RESULTS: Of 274 records identified, nine studies were eligible to be included. Despite the inconsistent observations in terms of diversity and richness, most studies reported alteration in bacterial communities between pre-cancer or cancer cases and control groups. The bacterial profile among cases was so far correlated at the phylum level with a noticeable diverse degree at the genus level. The majority of included studies reported a higher abundance of certain kinds of microorganisms as compared to healthy participants including Firmicutes, Fusobacteria and Actinobacteria at phyla level as well as Streptococcus, Actinomyces, Leptotrichia, Campylobacter, and Fusobacterium at the genus level. CONCLUSION: The alteration of the tongue microbial community has been associated with several diseases mainly cancer. So, the tongue microbiome may serve as a promising diagnostic tool or as a long-term monitor in precancerous or cancer cases.

Label-free quantitative proteomic analysis of the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis to identify protein features relevant in biofilm formation.

BACKGROUND: The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. This study investigated interactions between F. nucleatum and P. gingivalis proteomes with the objective to identify proteins relevant in biofilm formation. METHODS: We applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells grown in biofilm or planktonic culture, and as mono- and dual-species models. The detected proteins were classified into functional categories and their label-free quantitative (LFQ) intensities statistically compared. RESULTS: The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins, including abundant virulence factors. Using univariate statistics, we identified significant changes between biofilm and planktonic culture (p-value ≤0.05) in 0,4% F. nucleatum, 7% P. gingivalis, and 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolism had significantly increased levels in biofilm. In both mono- and dual-species biofilms, P. gingivalis increased the production of proteins for translation, oxidation-reduction, and amino acid metabolism compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. CONCLUSIONS: The findings suggest that P. gingivalis reduces the production of multiple proteins because of the F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilms, which need to be considered in the design of prevention strategies.

The effect of metronidazole plus amoxicillin or metronidazole plus penicillin V on periodontal pathogens in an in vitro biofilm model.

A combination of metronidazole (MET) and amoxicillin (AMX) is commonly used as adjunct to mechanical therapy of periodontal disease. The use of broad spectrum antibiotics such as AMX may contribute to development of antibiotic resistance. The aim was to evaluate the in vitro effect of replacing AMX with penicillin V (PV) in combination with MET on a biofilm model. A biofilm model consisting of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum was developed. The biofilms were exposed to AMX + MET and PV + MET in two different concentrations. Bacterial viability in biofilms following antibiotic exposure was assessed by viable counts and by confocal microscopy. No live colonies of P. gingivalis nor F. nucleatum were retrieved from biofilms exposed to AMX + MET or PV + MET. The amount of A. actinomycetemcomitans was 4-5 logs reduced following antibiotic treatment; no statistical significance was achieved between AMX + MET or PV + MET treated biofilms. Replacement of AMX with PV at the same concentration, in combination with MET, resulted in similar effect on bacterial viability in this in vitro model. The option of using PV + MET instead of AMX + MET deserves further investigation, as this may contribute to reduce the risk of antibiotic resistance development.

Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development.

Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K.

BACKGROUND: Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. METHODS: F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. RESULTS: Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. CONCLUSION: DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.