Supragingival Dental Plaque Research Articles

The effect of different sweeteners on the oral microbiome: a randomized clinical exploratory pilot study

ABSTRACT Introduction The aim of the study was to evaluate the modulating effects of five commonly used sweetener (glucose, inulin, isomaltulose, tagatose, trehalose) containing mouth rinses on the oral microbiome. Methods A single-centre, double-blind, parallel randomized clinical trial was performed with healthy, 18–55-year-old volunteers (N = 65), who rinsed thrice-daily for two weeks with a 10% solution of one of the allocated sweeteners. Microbiota composition of supragingival dental plaque and the tongue dorsum coating was analysed by 16S RNA gene amplicon sequencing of the V4 hypervariable region (Illumina MiSeq). As secondary outcomes, dental plaque red fluorescence and salivary pH were measured. Results Dental plaque microbiota changed significantly for two groups: inulin (F = 2.0239, p = 0.0006 PERMANOVA, Aitchison distance) and isomaltulose (F = 0.67, p = 0.0305). For the tongue microbiota, significant changes were observed for isomaltulose (F = 0.8382, p = 0.0452) and trehalose (F = 1.0119, p = 0.0098). In plaque, 13 species changed significantly for the inulin group, while for tongue coating, three species changed for the trehalose group (ALDEx2, p < 0.1). No significant changes were observed for the secondary outcomes. Conclusion The effects on the oral microbiota were sweetener dependant with the most pronounced effect on plaque microbiota. Inulin exhibited the strongest microbial modulating potential of the sweeteners tested. Further full-scale clinical studies are required.

A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism.

Calcifying nanoparticles (CNPs), referred to as nanobacteria (NB), are recognized to be associated with ectopic calcification. This study aims to isolate and culture CNPs from the dental plaque of patients with periodontal disease and investigate their possible role in unravelling the aetiology of periodontal disease. Supragingival and subgingival plaques were sampled from 30 periodontitis patients for CNPs isolation and culture. Alkaline phosphatase (ALP) content changes were tracked over time. Positive samples underwent thorough morphological identification via hematoxylin and eosin (HE) staining, Alizarin red S (ARS), and transmission electron microscopy (TEM). The chemical composition of CNPs analysis involved calcium (Ca) and phosphorus (P) content determination, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The subgingival plaque dental group exhibited a higher CNPs isolation rate at 36.67% (11/30) compared to the supragingival dental plaque group at 66.67% (20/30). ALP activity varied among the positive, negative and control groups. Morphological observation characterized the CNPs as round, oval, and ellipsoid particles with Ca deposits. Chemical analysis revealed the Ca/P ratio was 0.6753. Hydroxyl, methyl, carbonate, phosphate, hydrogen phosphate, and dihydrogen phosphate were detected by FTIR; the main chemical components detected by XRD were hydroxyapatite and tricalcium phosphate. CNPs were found in periodontitis-related dental plaque and exhibited the potential to develop calcified structures resembling dental calculus. However, the potential involvement of ALP in CNPs formation requires deeper exploration, as does the precise nature of its role and the interrelation with periodontitis demand a further comprehensive investigation.

Quantitative analysis of the effects of brushing, flossing, and mouthrinsing on supragingival and subgingival plaque microbiota: 12-week clinical trial

BackgroundTranslational microbiome research using next-generation DNA sequencing is challenging due to the semi-qualitative nature of relative abundance data. A novel method for quantitative analysis was applied in this 12-week clinical trial to understand the mechanical vs. chemotherapeutic actions of brushing, flossing, and mouthrinsing against the supragingival dental plaque microbiome. Enumeration of viable bacteria using vPCR was also applied on supragingival plaque for validation and on subgingival plaque to evaluate interventional effects below the gingival margin.MethodsSubjects with gingivitis were enrolled in a single center, examiner-blind, virtually supervised, parallel group controlled clinical trial. Subjects with gingivitis were randomized into brushing only (B); brushing and flossing (BF); brushing and rinsing with Listerine® Cool Mint® Antiseptic (BA); brushing and rinsing with Listerine® Cool Mint® Zero (BZ); or brushing, flossing, and rinsing with Listerine® Cool Mint® Zero (BFZ). All subjects brushed twice daily for 1 min with a sodium monofluorophosphate toothpaste and a soft-bristled toothbrush. Subjects who flossed used unflavored waxed dental floss once daily. Subjects assigned to mouthrinses rinsed twice daily. Plaque specimens were collected at the baseline visit and after 4 and 12 weeks of intervention. Bacterial cell number quantification was achieved by adding reference amounts of DNA controls to plaque samples prior to DNA extraction, followed by shallow shotgun metagenome sequencing.Results286 subjects completed the trial. The metagenomic data for supragingival plaque showed significant reductions in Shannon-Weaver diversity, species richness, and total and categorical bacterial abundances (commensal, gingivitis, and malodor) after 4 and 12 weeks for the BA, BZ, and BFZ groups compared to the B group, while no significant differences were observed between the B and BF groups. Supragingival plaque vPCR further validated these results, and subgingival plaque vPCR demonstrated significant efficacy for the BFZ intervention only.ConclusionsThis publication reports on a successful application of a quantitative method of microbiome analysis in a clinical trial demonstrating the sustained and superior efficacy of essential oil mouthrinses at controlling dental plaque compared to mechanical methods. The quantitative microbiological data in this trial also reinforce the safety and mechanism of action of EO mouthrinses against plaque microbial ecology and highlights the importance of elevating EO mouthrinsing as an integral part of an oral hygiene regimen.Trial registrationThe trial was registered on ClinicalTrials.gov on 31/10/2022. The registration number is NCT05600231.

Preliminary study on the diversity and quantity analysis of oral bacteria according to the sampling methods

Objectives: Oral bacterial samples included subgingival, supragingival, and saliva plaques. As the diversity and number of microorganisms deffer depending on the area of the oral cavity and the method used, an appropriate and reliable collection method is important. The present study investigated oral bacterial sampling methods. Methods: Supragingival dental plaque was collected from the buccal and lingual tooth surfaces of study participants using sterilized cotton swabs. Plaques were collected from the subgingival area using a sterilized curette. Bacterial genomic DNA was extracted using MagNA Pure 96 DNA and Viral NA low-volume kits. Real-time polymerase chain reaction (PCR) was performed using the PowerCheck™ Periodontitis Pathogens Multiplex Real-time PCR kit. Results: Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum of the orange complex were not observed in the subgingival biofilms of all study participants. For Porphyromonas. gingivalis, a significant correlation was observed between supragingival, subgingival, and total tooth surface biofilms. Compared to the supragingival and subgingival biofilmss, total tooth surface biofilm exhibited the highest bacterial count when the inswabbing method was used. Conclusions: Based on these findings, the supragingival swab method is recommended for oral bacterial research.

The use of an ultrasonic device in periodontal practice in elderly and senile people

The majority of periodontal visits in dental care to the population are made up of patients in the elderly age group (according to the WHO classification, these include persons 60–74 years old). Features of periodontal treatment of elderly people require a new approach to standard treatment regimens. In recent years, treatment with the «Vector» device (Dürr Dental, Germany) has found widespread use in periodontal consultations. The purpose of the study is to determine the volume of periodontal care for the elderly population in the State Medical Institution of the RO «SP» in Rostov-on-Don in dynamics from 2020 to 2022 using the «Vector» device. Results of the study: periodontal doctors performed ultrasonic removal of supragingival and subgingival dental plaque in the area of one tooth (using the Vector apparatus) in 2020, 1825 services, and in 2022 2562, which is 737 services more than in 2020, At the same time, 1241 services were performed for elderly people in 2020, and in 2022 there were 552 more services.Conclusion: the use of the «Vector» device in addition to traditional treatment regimens for chronic generalized periodontitis in elderly patients accelerates periodontal recovery time.

Association between oral health status and occlusal bite force in young adults

Background/purposeOral health is related to general health and a person’s overall well-being. The aim of the present study was to explore the association between oral health status and bite force among young adults. Materials and methodsMaximum bite force (MBF) was measured using Dental Prescale II in conjunction with a pressure-sensitive film and bite force analyzer in 40 young adults aged 20 to 40. Supragingival dental plaque was collected and cultured. Plaque weight, pH, and colony counts were assessed. The decayed, missing, and filled teeth index (DMFT) and body mass index (BMI) were recorded. ResultsBite force was negatively correlated with the number of missing teeth and the sum of missing and filled teeth. When the filled-to-remaining-teeth ratio (F/R ratio) was less than 8%, the bite force was significantly higher compared to an F/R ratio of 8–25%. Additionally, the amount of total bacteria was positively correlated with total bite force, and the quantity of Streptococcus mutans (S. mutans) along with total bacteria was positively correlated with bite force in the molar region (∗P < 0.05). The molar region predominantly contributed to bite force. ConclusionElevated levels of cariogenic bacteria may increase the risk of tooth loss, subsequently leading to reduced bite force. This reduction in bite force can further impact the efficiency of chewing function and, consequently, the quality of life. An F/R ratio above 8% could be easily calculated clinically and could serve as a guide to identify patients, particularly young adults, at risk of reduced bite force.

Abstract 2368: Pilot characterization of the salivary microbiome in pediatric cancer patients

Abstract Microbial populations in the oral cavity have been linked with some malignant diseases, however, the evidence in this regard is poor. Oral microorganisms increase cytokine production and other mediators may be related to carcinogenesis. However, the role of the oral microbiome and its taxonomic composition in pediatric cancer has not been studied. We report a pilot study to assess to compare the microbial diversity of saliva in pediatric patients with cancer and without cancer. Materials and methods: Total saliva samples were collected from 3 cases and 3 controls with cancer, with prior informed consent and assent. Metagenomic sequencing was performed and data was analyzed using CosmosID bioinformatics software. Results: the mean age for the cases is 8.67 years and for the 8-year-old controls, the percentage by sex was 33.3% male and 66.6% female. In the shared analysis of bacterial diversity we found a total of 93 genera, the three genera with the highest abundance in the cases were: Prevotella, Streptococcus and Morococcus. A total of 153 species with differential abundance were identified, in the cases the species with the highest abundance were: Neisseria mucosa, Vellodela dispar, Prevotella melaninogenica and Morococcus cerebrosus. We found differences in bacterial diversity and abundance between cases and controls. The main species that we found in greater abundance in the cases have been related to various processes in the oral cavity, Streptococcus sp increases child caries, Prevotella melaninogenica a broad profile of resistance to families of antibiotics, and Morococcus cerebrosus has been associated with the mineralization of the supragingival dental plaque. Finally Vellodela dispar has been associated with bacteremia in cancer patients. In this pilot study a small number of samples were analyzed, the differences in bacterial diversity should be confirmed by conducting studies with a larger number of samples. Citation Format: Alejandra Beatriz Cervantes Garduño, Milca de la O García, Jesús Adolfo Yamamoto Nagano, Rocio Del Socorro Cardenas Cardos, Liliana Velasco Hidalgo, Blanca Olivia Almazán García, Gabriela Elisa Mercado Celis. Pilot characterization of the salivary microbiome in pediatric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2368.

Streptococcus Mutans Serotype Analysis from Dental Plaque of Caries Patients in Banda Aceh Based on the GTF Gene.

Dental caries is an oral disease that is widely suffered by the population of Aceh caused by Streptococcus mutans. S. mutans serotypes c and d are widely isolated in the human oral cavity. This research was focused on detecting the presence and variability of S. mutans in supragingival dental plaque of caries teenager and young adults' patients. Subjects involved in this study were patients who treated at the Rumah Sakit Gigi dan Mulut of Dentistry Faculty of Universitas Syiah Kuala. The approach used in this research was molecular microbiology technique. To determine the presence of S. mutans, supragingival plaque from caries patients was cultivated in TYS20B. The culture findings were utilized to detect the presence of bacteria using PCR. The primers utilized in the PCR were S. mutans specific primers, GTFB (517 bp) for S. mutans serotype c and GTFI (712 bp) for S. mutans serotype d. Culture results on TYS20B media showed the growth of S. mutans colonies isolated from the supragingival plaque of research subjects. PCR results also revealed the presence of S. mutans in the supragingival plaques of caries patients, with the variability of S. mutans discovered to be a serotype c and a serotype d. Based on the findings of this study, it can be concluded that S. mutans can be found in the supragingival plaques of caries patients with the serotypes c and d variability.

Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.

Altered dental plaque microbiota correlated with salivary inflammation in female methamphetamine users.

Poor oral health is the most immediate and overlooked hazard of methamphetamine abuse in humans. Previous studies have reported methamphetamine-associated alterations in saliva microbiota, but the cause of methamphetamine-induced alterations in the oral microenvironment remains unclear. The present study aimed to investigate the alterations in dental plaque microbiota in methamphetamine users, and to explore their relationship with local immune inflammation in the oral cavity. This may provide new ideas on the development of methamphetamine-related oral microenvironment changes. Questionnaires and samples were obtained from 30 female methamphetamine users and 15 sex- and age-matched healthy controls. Microbial profiles of supragingival dental plaque were analyzed using 16S rRNA gene sequencing. Inflammatory factors in saliva were measured using enzyme-linked immunosorbent assay. Methamphetamine users had worse oral self-evaluation. Compared with healthy controls, methamphetamine users showed no differences in oral dental plaque microbial diversity but exhibited differences in the relative abundance of several microbial taxa. At the phylum level, a higher abundance of Proteobacteria and a lower abundance of Firmicutes were detected in methamphetamine users. Moreover, function prediction using the MetaCyc database showed that 33 pathways were significantly upregulated in methamphetamine users; Only the glycolytic (Pyrococcus) pathway was enriched in the C group. Importantly, salivary inflammatory factors showed complex significant associations with bacterial genera in methamphetamine users. Specifically, the genus Neisseria was positively correlated with IL-17 levels in saliva, and both were high in methamphetamine users. In contrast, the genus Streptococcus, with a lower abundance, was positively correlated with lower IL-10 levels. Overall, This study is the first to provide evidence for a link between altered dental plaque microbiota and salivary inflammation in methamphetamine users. Further elucidation of the interactions between methamphetamine use and oral microenvironment would be beneficial for appropriate interventions to improve oral health.

Evaluation of Association of Oral Bacterial Profile with HBV and HCV Infection and T Lymphocyte Level in HIV-Positive Patients.

Background This study was aimed to determine the oral bacterial profile of HIV-positive patients and their correlation with T lymphocyte and CD4 count and hepatitis B and C incidence. Methods In this study, 73 patients who were diagnosed HIV-positive and were referred to Shiraz HIV research center for routine dental treatment were enrolled. Demographic data including sex, ethnicity, CD4+ T cell, and T lymphocyte counts were collected from their medical records. Supragingival dental plaque and samples from the dorsal of the tongue were collected by sterile swabs. These samples were transferred to the microbiology laboratory of Jahrom University of Medical Sciences. After primary biochemical test of cultured samples, assessment of bacterial biofilms was done by DNA extraction. Real-time PCR with specific primer of each bacterial species was done, and assessment of the results of real time PCR led to determination of the species of the evaluated bacteria. The correlation of bacterial prevalence with hepatitis B and C was evaluated by chi-square test. Furthermore, Mann–Whitney test was used to evaluate the association of bacterial species prevalence with CD4 and T lymphocyte level. Results The prevalence of none of the detected bacteria had statistically significant relationship with hepatitis C, except for Peptostreptococcaceae (p value = 0.016) in the tongue plaque and Leptotrichia (p value = 0.022) in dental plaque. None of the evaluated bacteria showed any significant association with CD4 and T lymphocytes level, except for Kingella (p value = 0.025, 0.019, respectively), and also no significant correlation was reported with CD4, except for Gemella (p value = 0.021) and Campylobacter gracilis (p value = 0.029). Conclusions The diversity of the detected bacteria was more in dental plaque, while their density was more noticeable in the tongue plaque. No significant correlation was found between the prevalence of most of the detected bacteria and CD4 level and T lymphocyte level and incidence of hepatitis B and C.

Influence of Gallic Acid-Containing Mouth Spray on Dental Health and Oral Microbiota of Healthy Cats-A Pilot Study.

Simple SummaryPeriodontal diseases are common dental issues in cats. Oral care supplements were used to prevent diseases and maintain oral health. Moreover, maintaining a healthy oral microbiome is crucial for oral health. Therefore, we have developed a gallic acid-containing mouth spray and studied its effect on oral microbiota and dental health in healthy cats. The results revealed that the gingival and plaque indexes were improved after 42 days of mouth spray treatment in cats. The mouth spray treatment also reduced the abundance of harmful bacterial load and supported the growth of normal oral microbiota. This preliminary study recommended that the gallic acid-containing mouth spray could be an essential oral product to improve the oral hygiene of the cats.This pilot study aimed to investigate the effects of gallic acid-containing mouth spray on oral microbiota in healthy cat subjects. Forty healthy cats were recruited and randomly allocated to the control (G1; n = 20) and treatment groups (G2; n = 20). The cats were treated with mouth spray twice daily for 42 days. The changes in the gingival index (GI) and plaque index (PI) were measured at baseline (day 0) and end of the study (42nd day). The changes in the oral microbial composition of representative animals (control, n = 9; and treatment, n = 8) were also evaluated at baseline and end of the study. Oral microbial composition was assessed by amplifying the V1–V3 region of the 16S rRNA gene from supragingival dental plaque DNA extracts. The sequences were annotated using the QIIME 2.0. The GI and PI were significantly reduced after 42 days of treatment. The deep sequencing revealed that mouth spray influenced the cats’ oral microbiome and was significantly diverse. About 20 phyla and 59 species were observed after 42 days of mouth spray usage in cats’ oral microbiota. The number of operational taxonomic units (OTUs) of post-treatment samples (PoTS) of G2 was greatly reduced compared to other samples. Further analysis revealed that mouth spray acts substantially against Desulfomicrobium orale, one of the known pathogens in periodontal disease. The mouth spray efficiently reduced the growth of 22 species and uprooted 17 species. Moreover, the mouth spray supported the growth of normal oral microbiota, including Moraxella and Neisseria species. The preliminary study suggested that the gallic acids-containing mouth spray could be an essential oral product to improve the oral hygiene of the cats. Moreover, further studies are needed to confirm the beneficial effect of mouth spray on cats.

Molecular Detection of Streptococcus downii sp. nov. from Dental Plaque Samples from Patients with Down Syndrome and Non-Syndromic Individuals.

: BackgroundA new bacterial species has recently been identified in the dental plaque of an adolescent with Down syndrome. The species is known as Streptococcus downii sp. nov. (abbreviated to S. downii), and it inhibits the growth of S. mutans and certain periodontal pathogens. The aim of this study was to determine the distribution of S. downii in the oral cavity of individuals with Down syndrome. Methods: A specific polymerase chain reaction for the operon of bacteriocin (class IIb lactobin A/cerein 7B family) was designed to detect S. downii in individuals with Down syndrome (n = 200) and in the general population (n = 100). We also compared the whole genome of S. downii and the regions related to its bacteriocins against 127 metagenomes of supragingival plaque of the “Human Microbiome Project”. Results: We detected the specific gene of the S. downii bacteriocin in an individual with Down syndrome (Cq, 34.52; GE/μL, 13.0) and in an individual of the non-syndromic control group (Cq, 34.78 Cq; GE/μL, 4.93). The prevalence of S. downii was ≤1% both in Down syndrome and in the general population, which did not allow for clinical-microbiological correlations to be established. This result was confirmed by detecting only one metagenome with an ANIm with approximately 95% homology and with 100% homology with ORFs that code class IIb lactobiocin A/cerein 7B bacteriocins among the 127 metagenomes of the “Human Microbiome Project” tested. Conclusions: The detection rate of S. downii in the supragingival dental plaque was very low, both in the Down syndrome individuals and in the non-syndromic controls. A clinical-microbiological correlation could therefore not be established.

The sampling strategy of oral microbiome.

There are multiple habitats in the oral cavity with bacteria, fungi, viruses, and protozoaresiding in, which together constitute the oral micro-ecosystem. These microflorae in the oral cavity primarily include saliva, supragingival dental plaque, subgingival dental plaque, submucosal plaque around implants, plaque in root canals, and plaque on the mucosal surface. The interest and knowledge of the microbiome have dynamically increased with the advancement of technology. Therefore, a reliable, feasible, and practical sampling strategy for the oral microbiome is required for the investigation. This paper introduced the sampling strategy of oral microorganisms, consisting of sample collection, transport, processing, and storage. The materials and devices involved in this study are all commonly used in clinical practice or laboratory. The feasibility and reliability of the sampling methods described in this paper have been verified by multiple studies.

Streptococcus dentisani is a common inhabitant of the oral microbiota worldwide and is found at higher levels in caries-free individuals.

Streptococcus dentisani has been proposed as a promising probiotic against tooth decay, due to its ability to buffer acidic pH and to inhibit the growth of oral pathogens. However, it is unknown if this bacterial species has a global distribution. The current study aimed to establish the presence of S. dentisani in oral samples from different geographic locations by identifying the sequence of its 16S rRNA gene in available datasets from across the globe. In addition, an analytical and cross-sectional study was carried out to determine if the levels of this probiotic strain are higher in caries-free individuals compared to those with dental caries. Samples from various geographical sources demonstrated that S. dentisani is present in saliva and dental plaque from individuals of different continents. Typical S. dentisani levels in saliva ranged from 104 to 105 cells/ml and a total of 106-107 cells in dental plaque. Using real-time qPCR, S. dentisani was quantified from supragingival dental plaque of 25 caries-free and 29 caries-active individuals from a Mexican children population, where significantly higher proportions of S. dentisani were found in the caries-free group (p = 0.002). Finally, a negative correlation was found between caries levels (as measured by the dmft caries index) and the percentage of S. dentisani (p < 0.001). Thus, the current manuscript indicates that this species has a global distribution, can be found in saliva and dental plaque, and appears to be present in higher numbers in plaque samples from caries-free children.

Bacterial Biofilm Growth on Various Dental Stabilization Systems for Avulsed and Luxated Teeth

With the increased incidence of traumatic injuries and the advanced understanding of the periodontal and alveolar healing process, teeth splinting has become a common practice for stabilizing traumatized teeth. Consequently, several splinting materials and techniques have been introduced in the past few years. Despite the detrimental role of bacterial biofilm on healing, the level of biofilm development on these material surfaces has not been well investigated. Bacterial biofilms are severely detrimental for periodontal healing of avulsed and luxated teeth. Thus, biofilm growth becomes a critical factor in selecting the material of choice for dental splints. In this study, we aim to assess the level of oral biofilm growth on four different splinting systems: Ribbond©, orthodontic NiTi wire, monofilament fishing line, and Titanium Trauma Splint. A total of 72 extracted anterior teeth were divided into four groups. We splinted six rows of three teeth each per group. The teeth selected were caries-free and periodontitis-free at the time of extraction. To assess biofilm growth, a supragingival dental plaque sample was cultured and directly inoculated into all groups. After 7 days, bacterial growth was quantified by live/dead fluorescent microscopy assay and colony forming unit counts (CFU). Using one-way ANOVA and Bonferroni’s post hoc tests, we demonstrated that all splint systems allowed for bacterial growth. However, the Titanium Trauma Splint (TTS) allowed for the least amount of biofilm growth compared to other splint systems.

Bacterial Composition and Metabolomics of Dental Plaque From Adolescents.

Supragingival dental plaque samples were collected from 40 Swedish adolescents, including 20 with caries lesions (CAR) and 20 caries-free (CF). Fresh plaque samples were subjected to an ex vivo acid tolerance (AT) test where the proportion of bacteria resistant to an acid shock was evaluated through confocal microscopy and live/dead staining, and the metabolites produced were quantified by 1H Nuclear Magnetic Resonance (1H NMR). In addition, DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing, in order to characterize bacterial composition in the same samples. There were no significant differences in AT scores between CAR and CF individuals. However, 7 out of the 10 individuals with highest AT scores belonged to the CAR group. Regarding bacterial composition, Abiotrophia, Prevotella and Veillonella were found at significantly higher levels in CAR individuals (p=0.0085, 0.026 and 0.04 respectively) and Rothia and Corynebacterium at significantly higher levels in CF individuals (p=0.026 and 0.003). The caries pathogen Streptococcus mutans was found at low frequencies and was absent in 60% of CAR individuals. Random-forest predictive models indicate that at least 4 bacterial species or 9 genera are needed to distinguish CAR from CF adolescents. The metabolomic profile obtained by NMR showed a significant clustering of organic acids with specific bacteria in CAR and/or high AT individuals, being Scardovia wiggsiae the species with strongest associations. A significant clustering of ethanol and isopropanol with health-associated bacteria such as Rothia or Corynebacterium was also found. Accordingly, several relationships involving these compounds like the Ethanol : Lactate or Succinate : Lactate ratios were significantly associated to acid tolerance and could be of predictive value for caries risk. We therefore propose that future caries risk studies would benefit from considering not only the use of multiple organisms as potential microbial biomarkers, but also their functional adaptation and metabolic output.

ОЦЕНКА МАРКЕРОВ РАЗВИТИЯ ЗАБОЛЕВАНИЙ ПАРОДОНТА НА ОСНОВАНИИ ИЗУЧЕНИЯ МИКРОБНОГО СОСТАВА РАЗЛИЧНЫХ БИОТОПОВ ПОЛОСТИ РТА

Summary. The composition of supragingival and subgingival dental plaque, gingival and oral fluid, dentogingival or periodontal pocket, are a valuable diagnostic medium for non-invasive diagnosis of inflammatory periodontal diseases, since they contain a wide variety of microorganisms, many of which are markers.&#x0D; Purpose — non-invasive assessment of markers of periodontal diseases based on the study of the microbial composition of various biotopes of the oral cavity by PCR.&#x0D; Мaterials and methods. Non-invasive diagnosis risk factors, in particular, the presence of periodontal pathogenic microflora, provided for the study of various biotopes of the oral cavity — plaque, oral and gingival fluids, the composition of the dental pocket by applying a high-quality PCR diagnostic method using commercial DNA-express kits (LLC NPF «Litech», Russia).&#x0D; Results. A comparative study of markers of the development of periodontal diseases using a qualitative version of the PCR test system in samples of subgingival plaque and dental pocket contents of individuals with initial stage I periodontitis showed 3.1 times greater prevalence of the representative of the yellow complex — Aggregatibacter actinomycetemcomitans, 1.4 times more often than the representative of the red complex P. gingivalis. In the studied material of supragingival plaque, gingival and oral fluid of individuals with gingivitis induced by dental biofilm, the representative of the yellow complex Aggregatibacter actinomycetemcomitans prevails 2.6 times more often than representatives of the red complex — P. gingivalis and Tannerella forsythia, 1.4 times the orange complexFusobacterium nucleatum. In samples of supragingival plaque in individuals with clinically healthy gums with loss of periodontal tissue, representatives of the microflora that form orange, red and yellow complexes most often predominate.&#x0D; Conclusion. The study of supragingival and subgingival plaque, gingival and oral fluid, and the dentogingival pocket using a PCR test system allows timely detection of markers of the development of inflammatory diseases, regardless of clinical condition of periodontal disease.

In Vivo Colonization with Candidate Oral Probiotics Attenuates Colonization and Virulence of Streptococcus mutans.

A collection of 113 Streptococcus strains from supragingival dental plaque of caries-free individuals were recently tested in vitro for direct antagonism of the dental caries pathogen Streptococcus mutans, and for their capacity for arginine catabolism via the arginine deiminase system (ADS). To advance their evaluation as potential probiotics, twelve strains of commensal oral streptococci with various antagonistic and ADS potentials were assessed in a mouse model for oral (i.e., oral mucosal pellicles and saliva) and dental colonization under four diets (healthy or high-sucrose, with or without prebiotic arginine). Colonization by autochthonous bacteria was also monitored. One strain failed to colonize, whereas oral colonization by the other eleven strains varied by 3 log units. Dental colonization was high for five strains regardless of diet, six strains increased colonization with at least one high-sucrose diet, and added dietary arginine decreased dental colonization of two strains. Streptococcus sp. A12 (high in vitro ADS activity and antagonism) and two engineered mutants lacking the ADS (ΔarcADS) or pyruvate oxidase-mediated H2O2 production (ΔspxB) were tested for competition against S. mutans UA159. A12 wild type and ΔarcADS colonized only transiently, whereas ΔspxB persisted, but without altering oral or dental colonization by S. mutans In testing four additional candidates, S. sanguinis BCC23 markedly attenuated S. mutans' oral and dental colonization, enhanced colonization of autochthonous bacteria, and decreased severity of smooth surface caries under highly cariogenic conditions. Results demonstrate the utility of the mouse model to evaluate potential probiotics, revealing little correlation between in vitro antagonism and competitiveness against S. mutans in vivo IMPORTANCE Our results demonstrate in vivo testing of potential oral probiotics can be accomplished and can yield information to facilitate the ultimate design and optimization of novel anti-caries probiotics. We show human oral commensals associated with dental health are an important source of potential probiotics that may be used to colonize patients under dietary conditions of highly varying cariogenicity. Assessment of competitiveness against dental caries pathogen Streptococcus mutans and impact on caries identified strains or genetic elements for further study. Results also uncovered strains that enhanced oral and dental colonization by autochthonous bacteria when challenged with S. mutans, suggesting cooperative interactions for future elucidation. Distinguishing a rare strain that effectively compete with S. mutans under conditions that promote caries further validates our systematic approach to more critically evaluate probiotics for use in humans.

Oral Sampling Techniques.

The human oral cavity is a major point of entry for microorganisms, many of which live and multiply in the mouth. In addition, it provides an accessible site for sampling compared to other parts of the body; however, caution should be taken during oral sampling as many factors contribute to the microbial diversity in a site-dependent manner. The accessibility of the oral cavity and its microbial diversity emphasize the crucial need to avoid cross-contamination during the sampling procedure. In this chapter, we describe various detailed oral sampling procedures. These methods include supragingival dental plaque sampling, subgingival dental plaque sampling, oral mucosal sampling, and endodontic sampling methods for extracted teeth or in the patient's mouth. The proposed protocols provide tips to avoid contamination between different oral sources of bacteria and possible alternatives to the tools used.