A comparative study of microbial changes in dental plaque before and after single- and multiappointment treatments in patients with severe early childhood caries.

BACKGROUND: The status of dental caries is closely related to changes in the oral microbiome. In this study, we compared the diversity and structure of the dental plaque microbiome in children with severe early childhood caries (S-ECC) before and after general anaesthesia and outpatient treatment. METHODS: Forty children aged 3 to 5 years with S-ECC who had completed whole-mouth dental treatment under general anaesthesia (C1) or in outpatient settings (C2) were selected, 20 in each group. The basic information and oral health status of the children were recorded, and the microbial community structure and diversity of dental plaque before treatment (C1, C2), the day after treatment(C2_0D), 7 days after treatment (C1_7D, C2_7D), 1 month after treatment (C1_1M, C2_1M), and 3 months after treatment (C1_3M, C2_3M) were analysed via 16 S rRNA high-throughput sequencing technology. RESULTS: (1) The alpha diversity test showed that the flora richness in the multiappointment group was significantly greater at posttreatment than at pretreatment (P < 0.05), and the remaining alpha diversity index did not significantly differ between the 2 groups (P > 0.05). The beta diversity analysis revealed that the flora structures of the C1_7D group and the C2_3M group were significantly different from those of the other time points within the respective groups (P < 0.05). (2) The core flora existed in both the pre- and posttreatment groups, and the proportion of their flora abundance could be altered depending on the caries status of the children in both groups. Leptotrichia abundance was significantly (P < 0.05) lower at 7 days posttreatment in both the single- and multiappointment groups. Corynebacterium and Corynebacterium_matruchotii were significantly more abundant in the C1_1M and C1_3M groups than in the C1 and C1_7D groups (P < 0.05). Streptococcus, Haemophilus and Haemophilus_parainfluenzae were significantly more abundant in the C1_7D group than in the other groups (P < 0.05). CONCLUSION: A single session of treatment under general anaesthesia can cause dramatic changes in the microbial community structure and composition within 7 days after treatment, whereas treatment over multiple appointments may cause slow changes in oral flora diversity.

Analysis of Oral and Gut Microbiome Composition and Its Impact in Patients with Oral Squamous Cell Carcinoma.

The impact of gut and oral microbiota on the clinical outcomes of patients with oral squamous cell carcinoma (OSCC) is unknown. We compared the bacterial composition of dental plaque and feces between patients with OSCC and healthy controls (HCs). Fecal and dental plaque samples were collected from 7 HCs and 18 patients with OSCC before treatment initiation. Terminal restriction fragment-length polymorphism analysis of 16S rRNA genes was performed. Differences in bacterial diversity between the HC and OSCC groups were examined. We compared the occupancy of each bacterial species in samples taken from patients with OSCC and HCs and analyzed the correlation between PD-L1 expression in the tumor specimens and the occupancy of each bacterial species. The gut and oral microbiota of patients with OSCC were more varied than those of HCs. Porphyromonas and Prevotella were significantly more abundant in patients with OSCC than in HCs. The abundance of Clostridium subcluster XIVa in the gut microbiota of the PD-L1-positive group was significantly greater than that in the PD-L1-negative group. The oral and gut microbiomes of patients with OSCC were in a state of dysbiosis. Our results suggest the possibility of new cancer therapies targeting these disease-specific microbiomes using probiotics and synbiotics.

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

OBJECTIVES: 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. MATERIAL AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Species-level characterization of saliva and dental plaque microbiota reveals putative bacterial and functional biomarkers of periodontal diseases in dogs.

Periodontal diseases are among the most common bacterial-related pathologies affecting the oral cavity of dogs. Nevertheless, the canine oral ecosystem and its correlations with oral disease development are still far from being fully characterized. In this study, the species-level taxonomic composition of saliva and dental plaque microbiota of 30 healthy dogs was investigated through a shallow shotgun metagenomics approach. The obtained data allowed not only to define the most abundant and prevalent bacterial species of the oral microbiota in healthy dogs, including members of the genera Corynebacterium and Porphyromonas, but also to identify the presence of distinct compositional motifs in the two oral microniches as well as taxonomical differences between dental plaques collected from anterior and posterior teeth. Subsequently, the salivary and dental plaque microbiota of 18 dogs affected by chronic gingival inflammation and 18 dogs with periodontitis were compared to those obtained from the healthy dogs. This analysis allowed the identification of bacterial and metabolic biomarkers correlated with a specific clinical status, including members of the genera Porphyromonas and Fusobacterium as microbial biomarkers of a healthy and diseased oral status, respectively, and genes predicted to encode for metabolites with anti-inflammatory properties as metabolic biomarkers of a healthy status.

Supragingival dental biofilm profile and biofilm control during orthodontic treatment with fixed orthodontic appliance: A randomized controlled trial.

OBJECTIVE: The effectiveness of supragingival dental biofilm control during orthodontic treatment and changes in the bacterial profile were analyzed. DESIGN: Sixty-four participants aged 12-22 years (57% female) were included in the study. Participants underwent orthodontic treatment with fixed appliances and were randomly assigned to one of the three groups, which during a period of one month: (I) used chlorhexidine digluconate (CHX), (II) used high concentration of fluoride (F) gel and (III) performed standard oral hygiene. The plaque and gingivitis index, pH of biofilm and white spot lesions (WSL) were assessed. Changes of the bacteria in the biofilm were analyzed by the quantitative polymerase chain reaction RESULTS: Increase in the plaque index, pH of biofilm, and WSL was observed during orthodontic treatment with standard oral hygiene. Large interindividual variability was present, and the effects of one-month use of fluorides and CHX on clinical parameters were not significant. Despite standard hygiene the abundance of studied biofilm bacteria increased - the most Streptoccocus mutans (14.2x) and S. salivarius (3.3x), moderate Veillonella parvula (3x) and the least S. sobrinus (2.3x) and Agregatibacter actinomycetemcomitans (1.9x). The use of CHX reduced S. sobrinus (2.2x) and A. actinomycetemcomitans (1.9x). Fluoride use reduced A. actinomycetemcomitans (1.3x) and S. sobrinus (1.2x). Fluorides better controlled S. mutans than CHX. CONCLUSION: Bacterial biomass in supragingival biofilm increased during treatment with metal orthodontic appliances, with greater increase in cariogenic bacteria than periopathogens. Fluoride controlled S. mutans, while CHX S. sobrinus and A. actinomycetemcomitans.

Efficacy of a mouthwash containing ε-poly-L-lysine, funme peptides and domiphen in reducing halitosis and supragingival plaque: a randomized clinical trial.

OBJECTIVE: To evaluate the antibacterial effectiveness of a combination of ε-poly-L-lysine (ε-PL), funme peptide (FP) as well as domiphen against oral pathogens, and assess the efficacy of a BOP® mouthwash supplemented with this combination in reducing halitosis and supragingival plaque in a clinical trial. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans were determined by the gradient dilution method. Subsequently, the CCK-8 assay was used to detect the toxicity of mouthwash on human gingival fibroblastst, and the effectiveness in reducing halitosis and supragingival plaque of the mouthwash supplemented with the combination was analyzed by a randomized, double-blind, parallel-controlled clinical trial. RESULTS: The combination exhibited significant inhibitory effects on tested oral pathogens with the MIC < 1.56% (v/v) and the MBC < 3.13% (v/v), and the mouthwash containing this combination did not inhibit the viability of human gingival fibroblasts at the test concentrations. The clinical trial showed that the test group displayed notably lower volatile sulfur compounds (VSCs) at 0, 10, 24 h, and 7 d post-mouthwash (P < 0.05), compared with the baseline. After 7 days, the VSC levels of the and control groups were reduced by 50.27% and 32.12%, respectively, and notably cutting severe halitosis by 57.03% in the test group. Additionally, the Plaque Index (PLI) of the test and control group decreased by 54.55% and 8.38%, respectively, and there was a significant difference in PLI between the two groups after 7 days (P < 0.01). CONCLUSIONS: The combination of ε-PL, FP and domiphen demonstrated potent inhibitory and bactericidal effects against the tested oral pathogens, and the newly formulated mouthwash added with the combination exhibited anti-dental plaque and anti-halitosis properties in a clinical trial and was safe. TRIAL REGISTRATION: The randomized controlled clinical trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023).

Dental plaque microbiota sequence counts for microbial profiling and resistance genes detection.

Shotgun metagenomics sequencing experiments are finding a wide range of applications. Nonetheless, there are still limited guidelines regarding the number of sequences needed to acquire meaningful information for taxonomic profiling and antimicrobial resistance gene (ARG) identification. In this study, we explored this issue in the context of oral microbiota by sequencing with a very high number of sequences (~ 100 million), four human plaque samples, and one microbial community standard and by evaluating the performance of microbial identification and ARGs detection through a downsampling procedure. When investigating the impact of a decreasing number of sequences on quantitative taxonomic profiling in the microbial community standard datasets, we found some discrepancies in the identified microbial species and their abundances when compared to the expected ones. Such differences were consistent throughout downsampling, suggesting their link to taxonomic profiling methods limitations. Overall, results showed that the number of sequences has a great impact on metagenomic samples at the qualitative (i.e., presence/absence) level in terms of loss of information, especially in experiments having less than 40 million reads, whereas abundance estimation was minimally affected, with only slight variations observed in low-abundance species. The presence of ARGs was also assessed: a total of 133 ARGs were identified. Notably, 23% of them inconsistently resulted as present or absent across downsampling datasets of the same sample. Moreover, over half of ARGs were lost in datasets having less than 20 million reads. This study highlights the importance of carefully considering sequencing aspects and suggests some guidelines for designing shotgun metagenomics experiments with the final goal of maximizing oral microbiome analyses. Our findings suggest varying optimized sequence numbers according to different study aims: 40 million for microbiota profiling, 50 million for low-abundance species detection, and 20 million for ARG identification. KEY POINTS: • Forty million sequences are a cost-efficient solution for microbiota profiling • Fifty million sequences allow low-abundance species detection • Twenty million sequences are recommended for ARG identification.

Comparative characterization of supragingival plaque microbiomes in malocclusion adult female patients undergoing orthodontic treatment with removable aligners or fixed appliances: a descriptive cross-sectional study.

Objectives: This study aimed to explore the effects of removable aligners and fixed appliances on the supragingival bacterial communities in adult female patients undergoing orthodontic treatment. Methods: Supragingival plaque samples from 48 female individuals underwent microbiome analysis (16S rRNA gene sequencing) using PacBio Sequel sequencing. The study included 13 adults without orthodontic treatment needs as the control group (Group C), and 35 patients with comparable initial orthodontic conditions who received treatment at a university clinic in Beijing, China. The treatment involved either traditional fixed brackets (Group B, n = 17) or Invisalign® aligners (Group AT, n = 18). Bioinformatics methods were used for data analysis. Results: From the 48 plaque samples, a total of 334,961 valid reads were obtained, averaging 6,978 sequences per sample. The 16S rDNA sequences were classified into 25,727 amplicon sequence variants (ASVs). Significant variances in alpha and beta diversity among the groups were noted. Group B microbiome exhibited an increased presence of Gram-negative bacteria. At the phylum level, Actinobacteriota was significantly more prevalent in Group C samples, while Bacteroidota was enriched in Group B samples. Family-level relative abundance analysis showed a notable increase in Saccharibacteria (formerly TM7) and Prevotellaceae in Group B. Genus-level analysis revealed a significant rise in Lautropia in Group AT. Fixed orthodontic appliances were linked to oral microbiome changes, notably an enhanced relative abundance of anaerobes, including periodontal pathogens. Conclusion: The observation points to the impact of orthodontic appliance on the oral microbial community, highlighting the difference between traditional braces (Group B) and clear aligners (Group AT)in terms of the predominance of anaerobic and gram negative bacteria. This emphasizes the importance of considering the microbiological effects when choosing orthodontic appliance and underscores the need for tailored oral hygiene practices for individuals undergoing these treatments. This research might provide insights that could assist in the development of innovative cleaning techniques and antibacterial materials.

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

BACKGROUND: Translational 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. METHODS: Subjects 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. RESULTS: 286 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. CONCLUSIONS: This 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 REGISTRATION: The trial was registered on ClinicalTrials.gov on 31/10/2022. The registration number is NCT05600231.

Quantitative analysis of the effects of essential oil mouthrinses on clinical plaque microbiome: a parallel-group, randomized trial.

BACKGROUND: The rich diversity of microorganisms in the oral cavity plays an important role in the maintenance of oral health and development of detrimental oral health conditions. Beyond commonly used qualitative microbiome metrics, such as relative proportions or diversity, both the species-level identification and quantification of bacteria are key to understanding clinical disease associations. This study reports the first-time application of an absolute quantitative microbiome analysis using spiked DNA standards and shotgun metagenome sequencing to assess the efficacy and safety of product intervention on dental plaque microbiome. METHODS: In this parallel-group, randomized clinical trial, essential oil mouthrinses, including LISTERINE® Cool Mint Antiseptic (LCM), an alcohol-containing prototype mouthrinse (ACPM), and an alcohol-free prototype mouthrinse (AFPM), were compared against a hydroalcohol control rinse on clinical parameters and the oral microbiome of subjects with moderate gingivitis. To enable a sensitive and clinically meaningful measure of bacterial abundances, species were categorized according to their associations with oral conditions based on published literature and quantified using known amounts of spiked DNA standards. RESULTS: Multivariate analysis showed that both LCM and ACPM shifted the dysbiotic microbiome composition of subjects with gingivitis to a healthier state after 4 weeks of twice-daily use, resembling the composition of subjects with clinically healthy oral conditions recruited for observational reference comparison at baseline. The essential oil-containing mouthrinses evaluated in this study showed statistically significant reductions in clinical gingivitis and plaque measurements when compared to the hydroalcohol control rinse after 6 weeks of use. CONCLUSIONS: By establishing a novel quantitative method for microbiome analysis, this study sheds light on the mechanisms of LCM mouthrinse efficacy on oral microbial ecology, demonstrating that repeated usage non-selectively resets a gingivitis-like oral microbiome toward that of a healthy oral cavity. TRIAL REGISTRATION: The trial was registered on ClinicalTrials.gov on 10/06/2021. The registration number is NCT04921371.

Real-Time PCR Method as Diagnostic Tool for Detection of Periodontal Pathogens in Patients with Periodontitis.

The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.

Characterization and functional prediction of the dental plaque microbiome in patients with alveolar clefts.

Introduction: Alveolar cleft (AC) is a common congenital defect in people with cleft lip and palate (CLP). Alveolar bone grafting (ABG) is typically performed during adolescence, resulting in the fissure remaining in the mouth for a longer length of time. Patients with AC have a greater rate of oral diseases such as dental caries than the normal population, and the precise characteristics of the bacterial alterations caused by AC are unknown. Methods: We recruited a total of 87 subjects and collected dental plaque samples from AC adolescents (AAP), post-operative ABG adolescents (PAP), healthy control adolescents (CAP), AC young adults (AYP), post-operative ABG young adults (PYP), and healthy control young adults (CYP). The sequencing of 16S rRNA genes was performed. Results: The microbial composition of plaque from alveolar cleft patients differed significantly from age-matched healthy controls. Linear discriminant analysis effect size (LEfSe) analysis revealed that AAP was enriched for Neisseria, Haemophilus, Fusobacterium, Rhodococcus, Aggregatibacter, Gemella, and Porphyromonas, whereas AYP was enriched for Capnocytophaga, Rhodococcus, and Actinomyces-f0332. There were phenotypic differences in facultatively anaerobic, Gram-negative, Gram-positive, and oxidative stress tolerance between the AYP group with longer alveolar cleft and the healthy control group according to Bugbase phenotypic predictions. Alveolar bone grafting did not alter the functional phenotype of alveolar cleft patients but reduced the number of differential genera between alveolar cleft patients and healthy controls at both ages. Conclusions: Our study systematically characterized the supragingival plaque microbiota of alveolar cleft patients, post-alveolar bone grafting patients, and matched healthy controls in two ages to gain a better understanding of plaque ecology and microbiology associated with alveolar clefts.

Effects of toothpaste containing inactivated Lacticaseibacillus paracasei Probio-01 on plaque-induced gingivitis and dental plaque microbiota.

Plaque-induced gingivitis is an inflammatory response in gingival tissues resulting from bacterial plaque accumulation at the gingival margin. Postbiotics can promote the proliferation of beneficial bacteria and optimise the state of microbiota in the oral cavity. In this study, we investigated the effect of inactivated Lacticaseibacillus paracasei Probio-01 on plaque-induced gingivitis and the dental plaque microbiota. A total of 32 healthy gingival participants (Group N, using blank toothpaste for 3 months) and 60 patients with plaque-induced gingivitis (30 in Group F, using inactivated Probio-01 toothpaste for 3 months, and 30 in Group B, using blank toothpaste for 3 months, respectively) were recruited. Clinical indices, which included bleeding on probing (BOP), gingival index (GI), and plaque index (PI), were used to assess the severity of gingivitis. Furthermore, 16SrDNA amplicon sequencing was used to explore changes in the gingival state and dental plaque microbiota in patients with plaque-induced gingivitis. The results showed that inactivated Probio-01 significantly reduced clinical indices of gingivitis, including BOP, GI, and PI, in participants with plaque-induced gingivitis and effectively relieved gingival inflammation, compared with that observed in the control group (group B). Inactivated Probio-01 did not significantly influence the diversity of dental plaque microbiota, but increased the relative abundance of dental plaque core bacteria, such as Leptotrichia and Fusobacterium (P < 0.05). Strong correlations were observed between the indices and abundance of dental plaque microbiota. Overall, the inactivated Probio-01 significantly reduced the clinical indices of gingivitis and effectively improved gingival inflammation in patients with plaque-induced gingivitis. The activity of inactivated Probio-01 against plaque-induced gingivitis was possibly mediated by its ability to regulate the dental plaque microbiota, as indicated by the close correlation between the plaque microbiota and clinical indices of gingivitis.

Comparative analysis of microbial composition and functional characteristics in dental plaque and saliva of oral cancer patients.

BACKGROUND: The oral cavity is home to various ecological niches, each with its own unique microbial composition. Understanding the microbial communities and gene composition in different ecological niches within the oral cavity of oral cancer (OC) patients is crucial for determining how these microbial populations contribute to disease progression. METHODS: In this study, saliva and dental plaque samples were collected from patients with OC. Metagenomic sequencing was employed to analyze the microbial community classification and functional composition of the different sample groups. RESULTS: The results of the study revealed significant differences in both the function and classification of microbial communities between saliva and dental plaque samples. The diversity of microbial species in saliva was found to be higher compared to  that in plaque samples. Notably, Actinobacteria were enriched in the dental plaque of OC patients. Furthermore, the study identified several inter-group differential marker species, including Prevotella intermedia, Haemophilus parahaemolyticus, Actinomyces radius, Corynebacterium matruchitii, and Veillonella atypica. Additionally, 1,353 differential genes were annotated into 23 functional pathways. Interestingly, a significant correlation was observed between differentially labeled species and Herpes simplex virus 1 (HSV-1) infection, which may be related to the occurrence and development of cancer. CONCLUSIONS: Significant differences in the microbial and genetic composition of saliva and dental plaque samples were observed in OC patients. Furthermore, pathogenic bacteria associated with oral diseases were predominantly enriched in saliva. The identification of inter-group differential biomarkers and pathways provide insights into the relationship between oral microbiota and the occurrence and development of OC.

A comparison of the prevalence of respiratory pathogens and opportunistic respiratory pathogenic profile of 'clean' and 'unclean' removable dental prostheses.

OBJECTIVES: To determine and compare the opportunistic respiratory pathogenic index (ORPI) and prevalence of respiratory pathogens between clean and unclean removable prostheses. METHODS: A cross-sectional study was conducted among 97 removable prosthesis wearers at a teaching dental hospital. Participants' prosthesis hygiene was grouped into clean and unclean. After prosthesis plaque samples were sequenced using the Type IIB Restriction-site Associated DNA Sequencing for Microbiome method, the prevalence was assessed for the presence of respiratory pathogens on each sample. The ORPIs for clean and unclean prostheses were quantified based on the sum of the relative abundance of respiratory pathogenic bacteria in a microbiome using a reference database that contains opportunistic respiratory pathogens and disease-associated information. RESULTS: A total of 30 opportunistic respiratory pathogens were identified on the removable prostheses. Eighty-one (83.5 %) removable prostheses harboured respiratory pathogenic bacteria. Stenotrophomonas maltophilia (34.0 %), Pseudomonas aeruginosa (27.8 %), and Streptococcus agalactiae (27.8 %) were the top three prevalent respiratory pathogens detected in plaque samples. There was a significantly higher prevalence of respiratory pathogens residing on unclean than clean prostheses (P = 0.046). However, the ORPIs in both groups showed no statistically significant difference (P = 0.516). CONCLUSIONS: The ORPIs for both clean and unclean prostheses demonstrated a similar abundance of respiratory pathogens. However, the high prevalence of respiratory pathogens residing on unclean prostheses should not be underestimated. Therefore, maintaining good prosthesis hygiene is still important for overall oral and systemic health, even though the direct link between prosthesis cleanliness and reduced abundance of respiratory pathogens has not been established. CLINICAL SIGNIFICANCE: The association between the prevalence of respiratory pathogens and unclean removable prostheses has been demonstrated and might increase the theoretical risk of respiratory disease development.

Characteristics of the oral microbiota in patients with primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is an autoimmune disease with unknown etiology that is considered to be related to environmental and genetic factors. The aim of this study was to clarify the oral microflora characteristics of pSS patients and to reveal the connection between oral bacterial composition and dental caries using a high-throughput sequencing technique. METHODS: Thirty-five pSS patients and 20 healthy controls were enrolled in this study. We collected saliva and plaque samples from pSS patients and saliva samples from healthy controls. We used 16S ribosomal DNA (16S rDNA) high-throughput sequencing targeting the V3-V4 hypervariable region to determine the composition and structure of the microbiota in the three sample sets. Finally, bioinformatics analyses, including the diversity of the microbiota, species differences, and functional prediction were performed. RESULTS: In the alpha diversity and beta diversity analysis, the Chao1 (P < 0.01), observed species (P < 0.01), and PD whole tree indices (P < 0.01) were significantly lower in the saliva and plaque samples of pSS patients than in the saliva samples of healthy controls, but the Shannon (P < 0.01) and Simpson indices (P < 0.01) were significantly higher in the healthy controls, and their total diversity significantly differed. In the main flora composition at the genus level (top 10), we identified Prevotella and Veillonella as more enriched in the saliva of pSS patients and Fusobacterium, Actinomyces, and Leptotrichia as more enriched in the plaque of pSS patients. Predictive functional analysis showed that the oral microbiota of pSS patients was related to translation, metabolism of cofactors and vitamins, and nucleotide metabolism. CONCLUSIONS: The oral microbial ecology of patients with pSS is dysregulated, resulting in a decrease in overall diversity. Prevotella and Veillonella may be related to pSS, while Fusobacterium, Actinomyces, and Leptotrichia may be related to dental caries in pSS patients. Key Points • This study revealed differences in the oral microbial composition of patients with pSS compared to healthy controls. • We included a plaque group of pSS patients to identify the microbiota related to pSS and dental caries. • Prevotella and Veillonella may contribute to pSS, and Fusobacterium, Actinomyces, and Leptotrichia are associated with dental caries in pSS patients.

Microbiological Effects of Sodium Hypochlorite/-Amino Acids and Cross-linked Hyaluronic Acid Adjunctive to Non-surgical Periodontal Treatment.

PURPOSE: To investigate the microbiological outcomes obtained with either subgingival debridement (SD) in conjunction with a gel containing sodium hypochlorite and amino acids followed by subsequent application of a cross-linked hyaluronic acid gel (xHyA) gel, or with SD alone. MATERIALS AND METHODS: Forty-eight patients diagnosed with stages II-III (grades A/B) generalised periodontitis were randomly treated with either SD (control) or SD plus adjunctive sodium hypochlorite/amino acids and xHyA gel (test). Subgingival plaque samples were collected from the deepest site per quadrant in each patient at baseline and after 3 and 6 months. Pooled sample analysis was performed using a multiplex polymerase chain reaction (PCR)-based method for the identification of detection frequencies and changes in numbers of the following bacteria: Aggregatibacter actinomycetemcomitans (A.a), Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f), Treponema denticola (T.d), and Prevotella intermedia (P.i). RESULTS: In terms of detection frequency, in the test group, statistically significant reductions were found for P.g, T.f, T.d and P.i (p < 0.05) after 6 months. In the control group, the detection frequencies of all investigated bacterial species at 6 months were comparable to the baseline values (p > 0.05). The comparison of the test and control groups revealed statistically significant differences in detection frequency for P.g (p = 0.034), T.d (p < 0.01) and P.i (p = 0.02) after 6 months, favouring the test group. Regarding reduction in detection frequency scores, at 6 months, statistically significant differences in favour of the test group were observed for all investigated bacterial species: A.a (p = 0.028), P.g (p = 0.028), T.f (p = 0.004), T.d (p <0.001), and P.i (p = 0.003). CONCLUSIONS: The present microbiological results, which are related to short-term outcomes up to 6 months post-treatment, support the adjunctive subgingival application of sodium hypochlorite/amino acids and xHyA to subgingival debridement in the treatment of periodontitis.

Effects of Hydroxyapatite-Containing Toothpastes on Some Caries-Related Variables: A Randomised Clinical Trial.

OBJECTIVES: This randomised clinical trial was designed and carried out with the aim to evaluate the capacity of fluoride-substituted hydroxyapatite (HAF) toothpaste to modulate oral microflora composition and biofilm acidogenicity in schoolchildren. METHODS: In all, 610 children (4 to 5 and 6 to 7 years) were enrolled. Four toothpastes were randomly administered during 24 months: 2 contained fluoride-substituted hydroxyapatite (HAF1000 and HAF1450; 1000 and 1450 ppmF) and magnesium-, strontium-, and carbonate-substituted hydroxyapatite in a chitosan matrix, and 2 were monofluorophosphate fluoridated toothpastes (F1000 and F1450; 1000 and 1450 ppmF). Caries lesions were assessed by International Caries Detection and Assessment System scores, supragingival plaque was sampled from the approximal sites between primary molars using sterile Gracey curettes for microbiological analysis, and plaque pH curves after sucrose challenge were assessed at baseline and reevaluated after 1 year and after 2 years. The minimum and maximum pH decrease was calculated for caries-free patients and participants with a caries lesion(s) at baseline and at the end of the experimental period (24 months). Differences amongst measurements were analysed with 1-way analysis of variance. RESULTS: During the trial, the minimum pH value increased statistically significantly in all groups; in HAF1000 and HAF1450, the increase was greatest. At the end of trial, in the 2 HAF groups all primary cariogenic bacteria were statistically significantly lower with respect to F groups (P = .03 for Streptococcus mutans and sobrinus, for Lactobacillus casei, and for Lactobacillus fermentum). CONCLUSIONS: The trial provides robust but still inconclusive evidence on the efficacy of HAF toothpastes compared to traditional fluoridated toothpastes to reduce caries risk factors and to prevent caries lesions.

Psychological Stress and its relationship to Periodontal flora and salivary Nitrite/Nitrate.

OBJECTIVE: Psychological stress can be a common risk factor for the development of oral and systemic disease; therefore, analysis of a pathophysiologic mechanisms that may explain this association may be significant in planning preventive strategies. The aim of this study was to investigate the association amongst academic stress, periodontal health, and salivary cortisol and nitrite and nitrate levels in a sample of university students. METHODS: Participants (N = 14) were classified into 2 groups according to their exposure to academic stress due to periods of university exams (n = 6 and n = 8, respectively). All participants were subjected evlauted for their behavioural, psychological, and anthropometric parameters, as well as an oral health examination. A real-time polymerase chain reaction analysis in samples of saliva and plaque was used to detect Prevotella intermedia and Veillonella dispar as well as the total bacterial count. Nitrite/nitrate ratio (NR ratio) and cortisol in saliva were evaluated by enzyme-linked immunosorbent assay. RESULTS: Full Mouth Bleeding Score, Full Mouth Plaque Score, and Gingival Index were significantly higher in the group exposed to academic stress. Nitrite was directly related to the presence of V dispar (coefficient, 0.13; P = .00; CI, 0.07 to 0.19) and inversely related to total bacterial count (coefficient, -0.07; P = .012; CI, -0.13 to 0.02). NR ratio was directly related to V dispar (coefficient, 4.35; P = .010; 95% CI, 1.35 to 7.36) and inversely related to total bacterial count (coefficient, -4.05; P = .018; 95% CI, -7.32 to 0.86). CONCLUSIONS: These results confirm the importance of stress on periodontal health and salivary nitrite concentration and highlight a potential differential role of specific bacteria on nitrite concentration in saliva.

Effects of Sodium Hypochlorite Concentration and Application Time on Bacteria in an Ex Vivo Polymicrobial Biofilm Model.

INTRODUCTION: In endodontic treatment, it is important to remove or inactivate biofilms in the root canal system. We investigated the effects of different concentrations and application times of sodium hypochlorite (NaOCl) on the viability of bacteria in ex vivo polymicrobial biofilms of different maturation levels. METHODS: Polymicrobial biofilms were prepared from dental plaque samples and grown for 1, 2, and 3 weeks under anaerobic conditions on collagen-coated hydroxyapatite discs as an ex vivo biofilm model. The biofilms were then exposed to NaOCl at concentrations ranging from 0.1% to 2% for 1 or 3 minutes. The control group was exposed to sterile distilled water. Viability staining was performed and examined by confocal laser scanning microscopy to determine the percentage of biofilm bacteria killed by NaOCl. Scanning electron microscopy was also performed to visually examine the biofilms. RESULTS: Application of NaOCl at 0.5%-2% for both 1 and 3 min killed significantly more bacteria when compared to the controls (P < .05). Cell viability tended to be lower after the application of NaOCl for 3 minutes than that for 1 minute. CONCLUSIONS: Our experiments using an ex vivo model showed that within the range of 0.1%-2% of NaOCl, higher NaOCl concentrations and longer application times were more effective in killing biofilm bacteria, and that mature biofilms were more resistant to NaOCl than younger biofilms.