Unique miRomics Expression Profiles in Tannerella forsythia-Infected Mandibles during Periodontitis Using Machine Learning.

T. forsythia is a subgingival periodontal bacterium constituting the subgingival pathogenic polymicrobial milieu during periodontitis (PD). miRNAs play a pivotal role in maintaining periodontal tissue homeostasis at the transcriptional, post-transcriptional, and epigenetic levels. The aim of this study was to characterize the global microRNAs (miRNA, miR) expression kinetics in 8- and 16-week-old T. forsythia-infected C57BL/6J mouse mandibles and to identify the miRNA bacterial biomarkers of disease process at specific time points. We examined the differential expression (DE) of miRNAs in mouse mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels, which provided significant advantages over specific candidate miRNA or pathway analyses. All the T. forsythia-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, along with a significant increase in alveolar bone resorption (ABR) (p < 0.0001). We performed a NanoString analysis of specific miRNA signatures, miRNA target pathways, and gene network analysis. A total of 115 miRNAs were DE in the mandible tissue during 8 and 16 weeks The T. forsythia infection, compared with sham infection, and the majority (99) of DE miRNAs were downregulated. nCounter miRNA expression kinetics identified 67 downregulated miRNAs (e.g., miR-375, miR-200c, miR-200b, miR-34b-5p, miR-141) during an 8-week infection, whereas 16 upregulated miRNAs (e.g., miR-1902, miR-let-7c, miR-146a) and 32 downregulated miRNAs (e.g., miR-2135, miR-720, miR-376c) were identified during a 16-week infection. Two miRNAs, miR-375 and miR-200c, were highly downregulated with >twofold change during an 8-week infection. Six miRNAs in the 8-week infection (miR-200b, miR-141, miR-205, miR-423-3p, miR-141-3p, miR-34a-5p) and two miRNAs in the 16-week infection (miR-27a-3p, miR-15a-5p) that were downregulated have also been reported in the gingival tissue and saliva of periodontitis patients. This preclinical in vivo study identified T. forsythia-specific miRNAs (miR-let-7c, miR-210, miR-146a, miR-423-5p, miR-24, miR-218, miR-26b, miR-23a-3p) and these miRs have also been reported in the gingival tissues and saliva of periodontitis patients. Further, several DE miRNAs that are significantly upregulated (e.g., miR-101b, miR-218, miR-127, miR-24) are also associated with many systemic diseases such as atherosclerosis, Alzheimer's disease, rheumatoid arthritis, osteoarthritis, diabetes, obesity, and several cancers. In addition to DE analysis, we utilized the XGBoost (eXtreme Gradient boost) and Random Forest machine learning (ML) algorithms to assess the impact that the number of miRNA copies has on predicting whether a mouse is infected. XGBoost found that miR-339-5p was most predictive for mice infection at 16 weeks. miR-592-5p was most predictive for mice infection at 8 weeks and also when the 8-week and 16-week results were grouped together. Random Forest predicted miR-592 as most predictive at 8 weeks as well as the combined 8-week and 16-week results, but miR-423-5p was most predictive at 16 weeks. In conclusion, the expression levels of miR-375 and miR-200c family differed significantly during disease process, and these miRNAs establishes a link between T. forsythia and development of periodontitis genesis, offering new insights regarding the pathobiology of this bacterium.

Analysis of oral microbiome from fossil human remains revealed the significant differences in virulence factors of modern and ancient Tannerella forsythia.

BACKGROUND: Recent advances in the next-generation sequencing (NGS) allowed the metagenomic analyses of DNA from many different environments and sources, including thousands of years old skeletal remains. It has been shown that most of the DNA extracted from ancient samples is microbial. There are several reports demonstrating that the considerable fraction of extracted DNA belonged to the bacteria accompanying the studied individuals before their death. RESULTS: In this study we scanned 344 microbiomes from 1000- and 2000- year-old human teeth. The datasets originated from our previous studies on human ancient DNA (aDNA) and on microbial DNA accompanying human remains. We previously noticed that in many samples infection-related species have been identified, among them Tannerella forsythia, one of the most prevalent oral human pathogens. Samples containing sufficient amount of T. forsythia aDNA for a complete genome assembly were selected for thorough analyses. We confirmed that the T. forsythia-containing samples have higher amounts of the periodontitis-associated species than the control samples. Despites, other pathogens-derived aDNA was found in the tested samples it was too fragmented and damaged to allow any reasonable reconstruction of these bacteria genomes. The anthropological examination of ancient skulls from which the T. forsythia-containing samples were obtained revealed the pathogenic alveolar bone loss in tooth areas characteristic for advanced periodontitis. Finally, we analyzed the genetic material of ancient T. forsythia strains. As a result, we assembled four ancient T. forsythia genomes - one 2000- and three 1000- year-old. Their comparison with contemporary T. forsythia genomes revealed a lower genetic diversity within the four ancient strains than within contemporary strains. We also investigated the genes of T. forsythia virulence factors and found that several of them (KLIKK protease and bspA genes) differ significantly between ancient and modern bacteria. CONCLUSIONS: In summary, we showed that NGS screening of the ancient human microbiome is a valid approach for the identification of disease-associated microbes. Following this protocol, we provided a new set of information on the emergence, evolution and virulence factors of T. forsythia, the member of the oral dysbiotic microbiome.

Quantifying red complex bacteria, oral hygiene condition, and inflammation status in elderly: A pilot study.

Introduction: Periodontitis is an inflammation of the periodontal apparatus leads to destruction of connective tissue attachment and tooth loss. Red complex bacteria may contribute to disease initiation. Bacterial infection in periodontitis leads to a low-grade chronic infection and inflammation in distant organs. Notably, aging can affect the immune response. Objectives: The aim of this study was to analyze the effect of aging on oral hygiene and inflammation condition. Moreover, to evaluate the correlation between the oral hygiene condition and red complex bacterial load in subgingival plaque. Materials and methods: In this cross-sectional study, we examined 20 adult and 20 elderly subjects with periodontitis. Clinical parameters included Oral Hygiene Index Simplified (OHI-S) and Papillary Bleeding Index (PBI) were recorded. Subgingival plaque was collected from the tooth with a probing depth of 5-7 mm and analyzed with a reverse transcription polymerase chain reaction (RT-PCR) for red complex bacteria quantification. Statistical analysis was performed, respectively. Results: Both groups had poor oral hygiene conditions, reflected by high OHI-S and PBI. The quantity of red complex bacteria (P. gingivalis, T. denticola, T. forsythia) in the elderly group was significantly higher in comparison to the adult group. There was significant strong linear relationship between OHI-S and red complex bacteria (r < 1, p < 0.05). Only P. gingivalis bacteria with PBI values had a strong linear relationship and statistically significant. (r < 1, p < 0.05). P. gingivalis load was significantly higher than T. denticola and T. forsythia load, and it correlated with poor oral hygiene in the adult and elderly groups and with PBI in the elderly group. Conclusions: Aging affects to the red complex bacterial load and oral hygiene condition, but not the inflammation. These findings contribute to the development of novel treatment strategies focusing on bacterial aspect for periodontitis in the elderly.

Detection of Red complex bacteria, P. gingivalis, T. denticola and T. forsythia in infected root canals and their association with clinical signs and symptoms.

AIM: This study aimed to investigate the association between endodontic clinical signs and symptoms and the presence of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia employing polymerase chain reaction (PCR). MATERIALS AND METHODS: Microbial samples were obtained from 60 cases with necrotic pulp with primary teeth infections. DNA extracted from samples were analyzed for endodontic pathogens by using species-specific primers. RESULTS: P. gingivalis/T. denticola were detected in 15 symptomatic teeth associated with periapical lesions. T. forsythia/T. denticola were found in 16 symptomatic teeth associated with pain and swelling. P. gingivalis was detected in 9 teeth which were associated with pain, 2 with tenderness on percussion, and 15 with periapical lesions. Statistically significant associations were found between T. forsythia as well as T. denticola in relation to clinical findings of pain and swelling. (P < 0.05). Red complex bacteria showed no statistical significant association with the presence of signs and symptoms. CONCLUSION: Prevalence of P. gingivalis, T. denticola, and T. forsythia suggested association of these bacteria with symptomatic infected pulp and periradicular diseases.

Evaluation of role of periodontal pathogens in endodontic periodontal diseases.

AIM: This study aimed to correlate periodontal pathogens in endodontic periodontal diseases. METHODOLOGY: This study was conducted on 40 patients of both genders. All the participants were obtained from department of endodontics and periodontology with history of endo-perio lesion in same teeth. Polymerase chain reaction was performed and correlation was established. RESULTS: This study included 18 males and 22 females. The mean age of male was 42.5 years and female was 41.3 years. Specimens of Tannerella forsythia were isolated from 94% endodontium and 92% periodontium, Porphyromonas gingivalis from 71% endodontium and 55% periodontium, Aggregatibacter actinomycetemcomitans from 12% endodontium and 58% periodontium. The difference was significant (P < 0.05). Bacteria in endodontic-periodontal infection confirmed statistically significant correlation between absolute quantitation of T. forsythia and P. gingivalis (r = 0.412, P < 0.05), P. gingivalis and A. actinomycetemcomitans (r = 0.524, P < 0.05), and T. forsythia and A. actinomycetemcomitans (r = 0.427, P < 0.05). CONCLUSION: There was correlation between targeted bacterial species levels from concurrent endodontic-periodontal diseases. Thus, it can be suggested that dentinal tubules may be the pathway for spread of bacteria.

Detection of Tannerella forsythia bspA and prtH genotypes among periodontitis patients and healthy subjects-A case-Control study.

BACKGROUND: T. forsythia a gram negative, anaerobe inhabits the mature biofilm present at sites expressing progressive periodontitis. It is a part of "red complex" group which contributes to the pathogenesis of periodontitis. The BspA protein and prtH gene encoded cysteine protease play a vital role in the virulence of T. forsythia. The present study aims to detect the two genotypes (bspA and prtH) in periodontitis and healthy subjects. MATERIALS & METHOD: Subgingival plaque samples were collected from periodontitis patients and healthy subjects (Chronic Periodontitis n = 128, Aggressive Periodontitis n = 72, healthy subjects n = 200). The samples were screened for the presence of T. forsythia 16S rRNA, bspA and prtH genotypes by Polymerase Chain Reaction. The prevalence of the genotypes between periodontitis patients and healthy subjects was compared with Pearson's Chi-square test. A P value of < 0.05 was considered to be statistically significant. RESULTS: The prevalence for T. forsythia in Chronic Periodontitis (n = 128), Aggressive Periodontitis (n = 72) and health (n = 200) was 73.4%, 59.7% and 10.5% respectively. The prevalence of T.forsythia bspA/prtH genotypes was 81.90%/43.60%, 88.40%/53.50% and 33.30%/14.3% in Chronic Periodontitis, aggressive Periodontitis and health respectively. Compared to healthy subjects, the odds of detecting T.forsythia 16S rRNA was 18.53 times high in individuals with periodontitis (P = 0.0001). CONCLUSION: The high odds ratio of T.forsythia 16S rRNA among periodontitis strongly suggests its role in periodontitis. In addition, the high prevalence of T. forsythia bspA genotype among Chronic Periodontitis signifies it as a useful marker for chronic periodontitis.

Salivary ammonia levels and Tannerella forsythia are associated with rheumatoid arthritis: A cross sectional study.

The aim of this paper is to evaluate the relationship of salivary ammonium levels and the presence of bacteria with rheumatoid arthritis (RA) clinical disease activity in a cross-sectional study of Mexican patients. From a periodontal and disease activity standpoint, 132 consecutive RA patients fulfilling clinical criteria were evaluated. Ammonia levels (including peptidyl arginine deiminase activity) were evaluated by colorimetric assay and the presence of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia was evaluated by polymerase chain reaction (PCR) technique. After a multivariate analysis, adjusting for clinical and serological parameters, a significant association was only observed between severe periodontitis and probing depth with high RA disease activity. Additionally, in contrast to P. gingivalis, the presence of T. forsythia was significantly associated with high disease RA activity even after multivariable adjustment analysis. There was also a significant increase in ammonium levels in the high RA activity group and a significant correlation between salivary ammonia and RA disease activity but not with autoantibody titers. Similarly, we observed a significant increase in the ammonium levels derived from the cultures of P. gingivalis and T. forsythia, with respect to P. intermedia and S. gordonii cultures, or even healthy donors. These results suggest that RA activity is associated with severe periodontitis, high salivary ammonium levels and the presence of T. forsythia.

Characterization of an α-l-fucosidase from the periodontal pathogen Tannerella forsythia.

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 µM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase.