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.

Periodontitis exacerbates pulmonary hypertension by promoting IFNγ+ T cell infiltration in mice.

Uncovering the risk factors of pulmonary hypertension and its mechanisms is crucial for the prevention and treatment of the disease. In the current study, we showed that experimental periodontitis, which was established by ligation of molars followed by orally smearing subgingival plaques from patients with periodontitis, exacerbated hypoxia-induced pulmonary hypertension in mice. Mechanistically, periodontitis dysregulated the pulmonary microbiota by promoting ectopic colonization and enrichment of oral bacteria in the lungs, contributing to pulmonary infiltration of interferon gamma positive (IFNγ+) T cells and aggravating the progression of pulmonary hypertension. In addition, we identified Prevotella zoogleoformans as the critical periodontitis-associated bacterium driving the exacerbation of pulmonary hypertension by periodontitis, and the exacerbation was potently ameliorated by both cervical lymph node excision and IFNγ neutralizing antibodies. Our study suggests a proof of concept that the combined prevention and treatment of periodontitis and pulmonary hypertension are necessary.

Impact of Periodontitis on the Leakage of Oral Bacteria to the Gut.

Colorectal cancer (CRC) and periodontitis have recently been related due to the higher incidence of CRC in periodontal patients and the involvement of periodontal pathogens in carcinogenesis, suggesting that leakage from the oral cavity to the gut occurs. However, the magnitude of this pass-through in healthy individuals is controversial, and the effect that periodontitis could play in it is understudied. To evaluate the rate of bacterial leakage from the oral cavity to the gut, we analyzed the microbial composition of saliva, subgingival plaque, and fecal samples in healthy individuals without gastrointestinal disorders, including 20 periodontitis patients and 20 oral healthy controls, using PacBio full-length 16S rRNA gene sequencing. As expected, we observed a higher abundance of periodontal pathogens in the subgingival plaque and saliva of periodontal patients. In contrast, no significant differences were found between the fecal samples of both groups, implying that gut samples from periodontal patients were not enriched in periodontal pathogens. Fusobacterium nucleatum, a biomarker of CRC, was not found in the fecal samples of any participant. Our study does show a small leakage of some oral bacteria (mainly streptococci) to the gut, regardless of periodontal health status. Future studies should test whether other host factors and/or the preexistence of a gut disorder must be present in addition to periodontitis to promote the colonization of the gut by oral pathogens. The absence of periodontal pathogens in feces supports the idea that these bacteria could be used as biomarkers of intestinal disorders, including CRC.

Metagenomic analysis of healthy and diseased peri-implant microbiome under different periodontal conditions: a cross-sectional study.

BACKGROUND: Peri-implantitis is a polybacterial infection that can lead to the failure of dental implant rehabilitation. This study aimed to profile the microbiome of the peri-implant plaque and estimate the effect of periodontitis on it among 40 Chinese participants with dental implant prostheses and presenting with varying peri-implant and periodontal health states. METHODS: Submucosal plaque samples were collected from four distinct clinical categories based on both their implant and periodontal health status at sampling point. Clinical examinations of dental implant and remaining teeth were carried out. Metagenomic analysis was then performed. RESULTS: The microbiome of the peri-implantitis sites differed from that of healthy implant sites, both taxonomically and functionally. Moreover, the predominant species in peri-implantitis sites were slightly affected by the presence of periodontitis. T. forsythia, P. gingivalis, T. denticola, and P. endodontalis were consistently associated with peri-implantitis and inflammatory clinical parameters regardless of the presence of periodontitis. Prevotella spp. and P. endodontalis showed significant differences in the peri-implantitis cohorts under different periodontal conditions. The most distinguishing function between diseased and healthy implants is related to flagellar assembly, which plays an important role in epithelial cell invasion. CONCLUSIONS: The composition of the peri-implant microbiome varied in the diseased and healthy states of implants and is affected by individual periodontal conditions. Based on their correlations with clinical parameters, certain species are associated with disease and healthy implants. Flagellar assembly may play a vital role in the process of peri-implantitis.

Alterations and correlations in dental plaque microbial communities and metabolome characteristics in patients with caries, periodontitis, and comorbid diseases.

BACKGROUNDS: The pathogenic microorganisms and clinical manifestations of caries and periodontitis are different, caries and periodontitis are usually discussed separately, and the relationship between them is ignored. Clinically, patients prone to dental caries generally have a healthier periodontal status, whereas patients with periodontitis generally have a lower incidence of dental caries. The relationship between dental caries and periodontitis remains unclear. OBJECTIVES: This study aimed to explain the clinical phenomenon of antagonism between dental caries and periodontitis by exploring the ecological chain and bacterial interactions in dental caries, periodontitis, and other comorbid diseases. METHODS: The dental plaque microbiomes of 30 patients with oral diseases (10 each with caries, periodontitis, and comorbid diseases) were sequenced and analysed using 16 S rRNA gene sequencing. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) database was used for a differential functional analysis of dental plaque microbial communities in caries, periodontitis, and comorbid diseases. RESULTS: The coinfection group had the greatest bacterial richness in dental plaque. The principal coordinate analysis showed that caries and periodontitis were separate from each other, and comorbid diseases were located at the overlap of caries and periodontitis, with most of them being periodontitis. Simultaneously, we compared the microbiomes with significant differences among the three groups and the correlations between the microbiome samples. In addition, KEGG pathway analysis revealed significant differences in functional changes among the three groups. CONCLUSIONS: This study revealed the composition of the dental plaque microbial communities in caries, periodontitis, and comorbidities and the differences among the three. Additionally, we identified a possible antagonism between periodontitis and caries. We identified a new treatment strategy for the prediction and diagnosis of caries and periodontitis.

Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability.

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P < 0.05 in all five datasets with n = 20-82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P < 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P < 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies.

A distinctive subgingival microbiome in patients with periodontitis and Alzheimer's disease compared with cognitively unimpaired periodontitis patients.

AIM: Periodontitis is caused by dysbiosis of oral microbes and is associated with increased cognitive decline in Alzheimer's disease (AD), and recently, a potential functional link was proposed between oral microbes and AD. We compared the oral microbiomes of patients with or without AD to evaluate the association between oral microbes and AD in periodontitis. MATERIALS AND METHODS: Periodontitis patients with AD (n = 15) and cognitively unimpaired periodontitis patients (CU) (n = 14) were recruited for this study. Each patient underwent an oral examination and neuropsychological evaluation. Buccal, supragingival and subgingival plaque samples were collected, and microbiomes were analysed by next-generation sequencing. Alpha diversity, beta diversity, linear discriminant analysis effect size, analysis of variance-like differential expression analysis and network analysis were used to compare group oral microbiomes. RESULTS: All 29 participants had moderate to severe periodontitis. Group buccal and supragingival samples were indistinguishable, but subgingival samples demonstrated significant alpha and beta diversity differences. Differential analysis showed subgingival samples of the AD group had higher prevalence of Atopobium rimae, Dialister pneumosintes, Olsenella sp. HMT 807, Saccharibacteria (TM7) sp. HMT 348 and several species of Prevotella than the CU group. Furthermore, subgingival microbiome network analysis revealed a distinct, closely connected network in the AD group comprised of various Prevotella spp. and several anaerobic bacteria. CONCLUSIONS: A unique microbial composition was discovered in the subgingival region in the AD group. Specifically, potential periodontal pathogens were found to be more prevalent in the subgingival plaque samples of the AD group. These bacteria may possess a potential to worsen periodontitis and other systemic diseases. We recommend that AD patients receive regular, careful dental check-ups to ensure proper oral hygiene management.

Role of LL-37 in Oral Bacterial DNA Accumulation in Dental Plaque.

Dental plaque, a highly structured polymicrobial biofilm, persistently forms in the oral cavity and is a common problem affecting oral health. The role of oral defense factors in either collaborating or disrupting host-microbiome interactions remains insufficiently elucidated. This study aims to explore the role of LL-37, a critical antimicrobial peptide in the oral cavity, in dental plaque formation. Through immunostaining dental plaque specimens, we observed that LL-37 and DNA colocalized in the samples, appearing as condensed clusters. In vitro experiments revealed that LL-37 binds rapidly to oral bacterial DNA, forming high molecular weight, DNase-resistant complexes. This interaction results in LL-37 losing its inherent antibacterial activity. Further, upon the addition of LL-37, we observed a visible increase in the precipitation of bacterial DNA. We also discovered a significant correlation between the levels of the DNA-LL-37 complex and LL-37 within dental plaque specimens, demonstrating the ubiquity of the complex within the biofilm. By using immunostaining on dental plaque specimens, we could determine that the DNA-LL-37 complex was present as condensed clusters and small bacterial cell-like structures. This suggests that LL-37 immediately associates with the released bacterial DNA to form complexes that subsequently diffuse. We also demonstrated that the complexes exhibited similar Toll-like receptor 9-stimulating activities across different bacterial species, including Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius. However, these complexes prompted dissimilar activities, such as the production of IL-1ß in monocytic cells via both NLRP3 pathway-dependent and pathway-independent mechanisms. This study, therefore, reveals the adverse role of LL-37 in dental plaque, where it binds bacterial DNA to form complexes that may precipitate to behave like an extracellular matrix. Furthermore, the unveiled stimulating properties and species-dependent activities of the oral bacterial DNA-LL-37 complexes enrich our understanding of dental plaque pathogenicity and periodontal innate immune responses.

Changes in oral microflora following 0.3% cetylpyridinium chloride-containing mouth spray intervention in adult volunteers after professional oral care: Randomized clinical study.

OBJECTIVES: This study explored the changes in bacterial flora composition and total bacterial count in the saliva and tongue coating, along with the change in the tongue coating index (TCI) following an intervention with 0.3% cetylpyridinium chloride (CPC) mouth spray after professional oral care. MATERIALS AND METHODS: Fifty-two adult volunteers aged 30-60 years were equally divided into CPC spray (n = 26) and control (n = 26) groups. All subjects underwent scaling and polishing. The CPC spray group was administered four puffs of CPC spray to the tongue dorsum four times a day for 3 weeks. The control group performed only routine daily oral care (brushing) and did not use any other spray. Bacteriological evaluation of saliva and tongue coating was performed using 16S ribosomal RNA gene sequencing and quantitative polymerase chain reaction. The tongue coating was evaluated to calculate the TCI. A per-protocol analysis was conducted for 44 subjects (CPC spray group, n = 23; control group, n = 21). RESULTS: At 1 and 3 weeks after CPC spray use, the flora of the saliva and tongue coating changed; the genus Haemophilus was dominant in the CPC spray group, whereas the genus Saccharibacteria was dominant in the control group. The sampling time differed among individual participants, which may have affected the bacterial counts. There was no significant intragroup change in TCI in either group. CONCLUSIONS: CPC spray affected the bacterial flora in the saliva and tongue coating, particularly with respect to an increase in the abundance of Haemophilus. However, CPC spray did not change the TCI. These results suggest that it may be optimal to combine CPC spray with a physical cleaning method such as using a tongue brush or scraper. Clinical Trial Registration: University Hospital Medical Information Network UMIN000041140.

Factors affecting the number of bacteria in saliva and oral care methods for the recovery of bacteria in contaminated saliva after brushing: a randomized controlled trial.

BACKGROUND: Oral care is important in preventing aspiration pneumonia in older adults. However, it is not clear what kind of oral care can reduce the number of bacteria in saliva. The purposes of this study are to clarify whether there is a relationship between plaque amounts and salivary bacterial counts, and how bacteria dispersed into the oral cavity by brushing can be reduced. METHODS: First, saliva samples were collected from 10 healthy adult volunteers after 30 h of unbrushing and after thorough brushing, and the total bacterial count was determined by real-time PCR. Next, 40 older adults attending an outpatient dental clinic were randomly assigned into two groups: a wiping group (20 patients) and a mouthwashing group (20 patients). Saliva was collected before and after brushing, and after wiping in the wiping group and after mouthwashing in the mouthwashing group, and the total bacterial count was quantified by real-time PCR. RESULTS: In a study of volunteers, there was no association between plaque amounts and salivary bacterial counts. In a study of older adult patients, salivary bacterial counts were significantly higher in patients with higher oral hygiene index and fewer remaining teeth. Brushing increased salivary bacterial counts. Wiping did not significantly reduce the number of bacteria, while mouthwash returned the increased number of bacteria after brushing to the pre-brushing level. CONCLUSIONS: There is no direct relationship between the amount of plaque and the number of bacteria in saliva. Brushing disperses bacteria into the oral cavity, resulting in a marked increase in the number of bacteria in saliva. Wiping does not collect the dispersed bacteria, and it seems essential to rinse the mouth after brushing. TRIAL REGISTRATION: UMIN000045854.

New putative periodontopathogens and periodontal health-associated species: A systematic review and meta-analysis.

To investigate the existence of any association between new putative periodontal pathogens and periodontitis. Two independent reviewers conducted electronic literature searches in the MEDLINE (PubMed), EMBASE, DOSS and Google Scholar databases as well as a manual search to identify eligible clinical studies prior to November 2022. Studies comparing the prevalence of microorganisms other than the already-known periodontal pathogens in subgingival plaque and/or saliva samples between subjects with periodontitis and subject with periodontal health were included. Meta-analyses were performed on data provided by the included studies. Fifty studies including a total of 2739 periodontitis subjects and 1747 subjects with periodontal health were included. The Archaea domain and 25 bacterial species (Anaeroglobus geminatus, Bacteroidales [G-2] bacterium HMT 274, Desulfobulbus sp. HMT 041, Dialister invisus, Dialister pneumosintes, Eubacterium brachy, Enterococcus faecalis, Eubacterium nodatum, Eubacterium saphenum, Filifactor alocis, Fretibacterium sp. HMT 360, Fretibacterium sp. HMT 362, Mogibacterium timidum, Peptoniphilaceae sp. HMT 113, Peptostreptococcus stomatis, Porphyromonas endodontalis, Slackia exigua, Streptococcus gordonii, Selenomonas sputigena, Treponema amylovorum, Treponema lecithinolyticum, Treponema maltophilum, Treponema medium, Treponema parvum and Treponema socranskii) were found to be statistically significantly associated with periodontitis. Network studies should be conducted to investigate the role of these newly identified periodontitis-associated microorganisms through interspecies interaction and host-microbe crosstalk analyses.

The Oral Microbiome and Cross-Kingdom Interactions during Pregnancy.

Pregnancy initiates a temporary transition in the maternal physiological state, with a shift in the oral microbiome and a potential increase in frequency of oral diseases. The risk of oral disease is higher among populations of Hispanic and Black women and those with lower socioeconomic status (low SES), demonstrating a need for intervention within these high-risk populations. To further our understanding of the oral microbiome of high-risk pregnant women, we characterized the oral microbiome in 28 nonpregnant and 179 pregnant low-SES women during their third trimester living in Rochester, New York. Unstimulated saliva and supragingival plaque samples were collected cross-sectionally, followed by assessment of the bacterial (16S ribosomal RNA) and fungal (18S ITS) microbiota communities. Trained and calibrated dentists performed oral examinations to determine the number of decayed teeth and plaque index. Initially, plaque from 28 nonpregnant women and 48 pregnant women were compared; these data showed significant differences in bacterial abundances based on pregnancy status. To further our understanding of the oral microbiome within the pregnant population, we next examined the oral microbiome within this population based on several variables. Streptococcus mutans, Streptococcus oralis, and Lactobacillus were associated with a greater number of decayed teeth. The composition of fungal communities differed between plaque and saliva, demonstrating 2 distinct "mycotypes" that were represented by a greater abundance of Candida in plaque and Malassezia in saliva. Veillonella rogosae, a common oral bacterium, was negatively associated with both plaque index and salivary Candida albicans colonization by culture data. This was further emphasized by in vitro inhibition of C. albicans by V. rogosae. Identification of interactions between the bacterial or fungal oral communities revealed that V. rogosae was positively associated with the oral commensal Streptococcus australis and negatively with the cariogenic Lactobacillus genus, suggesting V. rogosae as a potential biomarker of a noncariogenic oral microbiome.

Chapter 11: Future Perspectives in the Study of Dental Caries.

Dental caries is a disease that affects people of all ages since demineralization and remineralization of tooth surfaces occur in everyone's mouths, and caries lesions develop when there is an imbalance between demineralization and remineralization. In this way, teeth are exposed to a risk of caries. Prevention strategies aiming at "zero caries" and treatments aiming at "tooth recovery and regeneration" are the two main areas of caries research, and both basic and clinical research are required in these fields. The following future perspectives of caries research were identified: The disease concept of caries is undergoing rapid structural changes, as it will increasingly become a disease of all generations: Changes in our understanding of caries etiology (from cariogenic pathogens in the specific plaque hypothesis to the oral microbiome in the ecological plaque hypothesis) will alter the concept of caries-associated bacteria (from mutans streptococci to a group of bacteria with high acid-producing capacity and acid-tolerance or acidogenic/aciduric bacteria). In the field of prevention, more individualized, site-specific, and high-precision examinations for risk assessment and diagnostic methods, including genetic tests, will be developed, and advanced preventive, curative, and regenerative treatments will become possible. To achieve this, interdisciplinary, multidisciplinary, and transdisciplinary research are essential, and collaboration and fusion with other sciences, such as material science, engineering, food science, and nutritional science, are required. Furthermore, in order to put the results of such research into practice in society, it will be necessary to promote industry-academia collaborations; promote behavioral change through sociological approaches; and correct economic, informational, and educational inequalities. The sociological approach requires the coupling of epidemiology and data science as well as the validation of clinical applications, and artificial intelligence will play a powerful role in such analyses.

Patients with type 2 diabetes and severe periodontitis harbor a less pathogenic subgingival biofilm than normoglycemic individuals with severe periodontitis.

BACKGROUND: Whether, and to what extent, diabetes mellitus (DM) can affect the subgingival biofilm composition remains controversial. Thus, the aim of this study was to compare the composition of the subgingival microbiota of non-diabetic and type 2 diabetic patients with periodontitis using 40 "biomarker bacterial species." METHODS: Biofilm samples of shallow (probing depth [PD] and clinical attachment level [CAL] ≤3 mm without bleeding) and deep sites (PD and CAL ≥5 mm with bleeding) of patients with or without type 2 DM were evaluated for levels/proportions of 40 bacterial species by checkerboard DNA-DNA hybridization. RESULTS: A total of 828 subgingival biofilm samples from 207 patients with periodontitis (118 normoglycemic and 89 with type 2 DM) were analyzed. The levels of most of the bacterial species evaluated were reduced in the diabetic compared with the normoglycemic group, both in shallow and in deep sites. The shallow and deep sites of patients with type 2 DM presented higher proportions of Actinomyces species, purple and green complexes, and lower proportions of red complex pathogens than those of normoglycemic patients (P < 0.05). CONCLUSIONS: Patients with type 2 DM have a less dysbiotic subgingival microbial profile than normoglycemic patients, including lower levels/proportions of pathogens and higher levels/proportions of host-compatible species. Thus, type 2 diabetic patients seem to require less remarkable changes in biofilm composition than non-diabetic patients to develop the same pattern of periodontitis.

Symbiotic relationship between Prevotella denticola and Streptococcus mutans enhances virulence of plaque biofilms.

OBJECTIVES: This study aimed to explore that whether interactions between Prevotella denticola and Streptococcus mutans could promote the establishment of hypervirulent biofilms on teeth surface and eventually influence the occurrence and development of caries. DESIGN: Based on single-species biofilms of either P. denticola or S. mutans, and dual-species biofilms of both bacteria, we compared the virulence properties associated with cariogenicity in vitro, including carbohydrate metabolism and acid productivity, synthesis of extracellular polysaccharides, biomass and architecture of biofilms, level of enamel demineralization and expression of virulence genes associated with carbohydrate metabolism and adhesion in S. mutans. RESULTS: The data demonstrated that, compared to single-species of above two taxa, dual-species produced lactate by metabolizing carbohydrates at a higher level during the observation period. Moreover, dual-species biofilms accrued more biomass and exhibited more dense microcolonies and abundant extracellular matrix. And it's noticeable that the level of enamel demineralization in dual-species biofilms was more augmented than that of single-species. In addition, the presence of P. denticola induced the expression of virulence genes gtfs and gbpB in S. mutans. CONCLUSIONS: Symbiotic relationship between P. denticola and S. mutans enhances caries-associated virulence of plaque biofilms, which might provide new strategies for effective prevention and treatment of caries.

Comparative 16S rRNA gene sequencing study of subgingival microbiota of healthy subjects and patients with periodontitis from four different countries.

AIM: To investigate the differences between the subgingival microbiota of healthy subjects (HS) and periodontitis patients (PP) from four different countries through a metagenomic approach. MATERIALS AND METHODS: Subgingival samples were obtained from subjects from four different countries. Microbial composition was analysed through high-throughput sequencing of the V3-V4 region of the 16S rRNA gene. The country of origin, diagnosis and clinical and demographic variables of the subjects were used to analyse the microbial profiles. RESULTS: In total, 506 subgingival samples were analysed: 196 from HS and 310 from patients with periodontitis. Differences in richness, diversity and microbial composition were observed when comparing samples pertaining to different countries of origin and different subject diagnoses. Clinical variables, such as bleeding on probing, did not significantly affect the bacterial composition of the samples. A highly conserved core of microbiota associated with periodontitis was detected, while the microbiota associated with periodontally HS was much more diverse. CONCLUSIONS: Periodontal diagnosis of the subjects was the main variable explaining the composition of the microbiota in the subgingival niche. Nevertheless, the country of origin also had a significant impact on the microbiota and is therefore an important factor to consider when describing subgingival bacterial communities.

Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT).

OBJECTIVE: The aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome. DESIGN: Total nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis. RESULTS: The automated extraction method at 63°C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363. CONCLUSIONS: In general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites.

High-Resolution Detection of Translocation of Oral Bacteria to the Gut.

Ectopic enrichment of oral microbes in the gut is a notable alteration in gut microbial balance. These microbes are likely delivered from the oral cavity with saliva and food; however, evidence of oral-gut microbial transmission is insufficient and needs further investigation. In this observational study, we examined 144 pairs of saliva and stool samples collected from community-dwelling adults to verify the oral-gut microbial link and identify the relevant influencing factors on the increased abundance of oral microbes within the gut. The bacterial composition of each sample was determined using PacBio single-molecule long-read sequencing of the full-length 16S ribosomal RNA gene and amplicon sequence variant (ASV) analysis. Although the bacterial compositions of salivary and gut microbiota were distinctly different, at least 1 ASV was shared between salivary and gut microbiota in 72.9% of subjects. Shared ASVs accounted for 0.0% to 63.1% (median 0.14%) of the gut microbiota in each subject and frequently included abundant Streptococcus salivarius and Streptococcus parasanguinis. Their total relative abundance in the gut was significantly higher in older subjects or those with dental plaque accumulation. The gut microbiota with ≥5% of shared ASVs displayed a higher abundance of Streptococcus, Lactobacillus, and Klebsiella and a lower abundance of Faecalibacterium, Blautia, Megamonas, and Parabacteroides. Our study presents evidence for the translocation of oral bacteria to the gut in community-dwelling adults and suggests that aging and dental plaque accumulation contribute to an increased abundance of oral microbes in the gut, which might be relevant to the compositional shift in the gut commensals.

The association between Aggregatibacter actinomycetemcomitans JP2 clone and periodontitis: A systematic review and meta-analysis.

To appraise the literature on the prevalence of the JP2 clone of Aggregatibacter actinomycetemcomitans (A.a.) and on its association with presence and progression of periodontitis in different populations. A systematic search of the literature was conducted in Medline, Embase and Cochrane Library for studies reporting data on detection of the JP2 clone of A.a. A total of 56 papers were included in the review, from an initial search of 685 titles. Studies were carried out in populations with a mean age of 26.34 years (range 6.24-53.85 years). Just over 16% of the overall population assessed (n = 13 751) had the JP2 clone detected. Meta-analyses included 16 studies and 1775 patients, and revealed an association between detection of the JP2 clone and diagnosis of periodontitis (RR = 1.86, 95% 1.43-2.42) from saliva and plaque, with high heterogeneity (I2  = 85%, p < .00001). Meta-analyses included 5 studies and 616 patients, and revealed an association between baseline detection of the JP2 clone and onset of periodontitis over 2 to 5 years (RR = 4.12, 95% 2.42-7.00), with high heterogeneity (I2  = 81%, p < .0003). From the overall risk of bias score, 29 papers were judged as low risk of bias, whilst the remaining papers were judged to have an overall medium or high risk of bias. Detection of the JP2 clone of A.a. in subgingival plaque and saliva samples is associated with increased odds of diagnosis of periodontitis and may be able to predict onset of periodontitis. This systematic review provides clear evidence that in certain populations, the JP2 clone of A.a. is associated with early-onset periodontitis. Furthermore, detection of this bacterium seems to be predictive of disease onset.

Longitudinal changes in subgingival biofilm composition following periodontal treatment.

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