Linking periodontal pathogens with endoperio lesions.

Endodontic-periodontal diseases pose difficulties for the practitioner in diagnosing and predicting the success of the affected teeth. Therefore, it is of interest to correlate between periodontal infections and endodontic periodontal disorders. 50 patients of both sexes were included in this study. 28 of the 50 patients were men and 22 were women. Participants with a history of endodontic and periodontal lesions on the same tooth were chosen. A polymerase chain reaction experiment was carried out and relationships were formed. Data shows that isolates of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Tannerella forsythia were identified in 91% of the periodontium, 12% of the endodontium, and 51% of the endodontium, respectively. Targeted bacterial species were associated with periodontal and endodontic disorders that occurred concurrently. Therefore, it is plausible to speculate that dentinal tubules serve as a channel for the dissemination of microorganisms.

Porphyromonas endodontalis HmuY differentially participates in heme acquisition compared to the Porphyromonas gingivalis and Tannerella forsythia hemophore-like proteins.

Introduction: Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein. Methods: Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium. Results: Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin. Conclusions: We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.

The intriguing strategies of Tannerella forsythia's host interaction.

Tannerella forsythia, a member of the "red complex" bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O-glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia, including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management.

Lung abscess caused by the anaerobic pathogen Tannerella forsythia.

Odontogenic infections can spread to the respiratory tract. Despite the known role of Tannerella forsythia as the primary pathogen in periodontitis, the association between T. forsythia infection and risk of pneumonia or lung abscess remains unknown. In this report, we present a case of lung abscess caused by T. forsythia infection. The pathogen was detected by metagenomic next-generation sequencing (mNGS) in the bronchoalveolar lavage fluid of the patient. The clinical characteristics and possible mechanisms of the infection are discussed. T. forsythia is a conditional pathogen that can cause lung abscess in the presence of helper bacteria and reduced host immune status. The course of treatment should be personalized and might be longer than 3 months.

Red-complex bacteria exhibit distinctly different interactions with human periodontal ligament stromal cells compared to Fusobacterium nucleatum.

OBJECTIVE: The red-complex bacteria Porphyromonas gingivalis and Tannerella forsythia together with Fusobacterium nucleatum are essential players in periodontitis. This study investigated the bacterial interplay with human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) which act in the acute phase of periodontal infection. DESIGN: The capability of the bacteria to induce an inflammatory response as well as their viability, cellular adhesion and invasion were analyzed upon mono- and co-infections of hPDL-MSCs to delineate potential synergistic or antagonistic effects. The expression level and concentration of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were measured using qRT-PCR and ELISA. Viability, invasion, and adhesion were determined quantitatively using agar plate culture and qualitatively by confocal microscopy. RESULTS: Viability of P. gingivalis and T. forsythia but not F. nucleatum was preserved in the presence of hPDL-MSCs, even in an oxygenated environment. F. nucleatum significantly increased the expression and concentration of IL-6, IL-8 and MCP-1 in hPDL-MSCs, while T. forsythia and P. gingivalis caused only a minimal inflammatory response. Co-infections in different combinations had no effect on the inflammatory response. Moreover, P. gingivalis mitigated the increase in cytokine levels elicited by F. nucleatum. Both red-complex bacteria adhered to and invaded hPDL-MSCs in greater numbers than F. nucleatum, with only a minor effect of co-infections. CONCLUSIONS: Oral bacteria of different pathogenicity status interact differently with hPDL-MSCs. The data support P. gingivalis' capability to manipulate the inflammatory host response. Further research is necessary to obtain a comprehensive picture of the role of hPDL-MSCs in more complex oral biofilms.

Ancient Genomes From Bronze Age Remains Reveal Deep Diversity and Recent Adaptive Episodes for Human Oral Pathobionts.

Ancient microbial genomes can illuminate pathobiont evolution across millenia, with teeth providing a rich substrate. However, the characterization of prehistoric oral pathobiont diversity is limited. In Europe, only preagricultural genomes have been subject to phylogenetic analysis, with none compared to more recent archaeological periods. Here, we report well-preserved microbiomes from two 4,000-year-old teeth from an Irish limestone cave. These contained bacteria implicated in periodontitis, as well as Streptococcus mutans, the major cause of caries and rare in the ancient genomic record. Despite deriving from the same individual, these teeth produced divergent Tannerella forsythia genomes, indicating higher levels of strain diversity in prehistoric populations. We find evidence of microbiome dysbiosis, with a disproportionate quantity of S. mutans sequences relative to other oral streptococci. This high abundance allowed for metagenomic assembly, resulting in its first reported ancient genome. Phylogenetic analysis indicates major postmedieval population expansions for both species, highlighting the inordinate impact of recent dietary changes. In T. forsythia, this expansion is associated with the replacement of older lineages, possibly reflecting a genome-wide selective sweep. Accordingly, we see dramatic changes in T. forsythia's virulence repertoire across this period. S. mutans shows a contrasting pattern, with deeply divergent lineages persisting in modern populations. This may be due to its highly recombining nature, allowing for maintenance of diversity through selective episodes. Nonetheless, an explosion in recent coalescences and significantly shorter branch lengths separating bacteriocin-carrying strains indicate major changes in S. mutans demography and function coinciding with sugar popularization during the industrial period.

Molecular Characterization of the glyA Gene From the Clinical Isolates of Tannerella forsythia.

BACKGROUND: The glyA gene in Tannerella forsythia is attributed for its virulence by producing the enzyme serine hydroxymethyltransferase (SHMT), which plays a vital role in bacterial cell metabolism. OBJECTIVES: The study is thus aimed to determine the frequency of the glyA gene from the clinical strains of T. forsythia isolated from periodontitis patients. MATERIALS AND METHODS: Forty-five patients with varying degrees of periodontitis were included in the study, and the plaque samples collected from them were anaerobically processed by inoculating onto sterile anaerobic blood agar plates using a gaspak system, with incubation at 37°C for 5-7 days. The DNA was extracted from the obtained isolated colony, and PCR was performed to confirm the presence of the glyA gene. RESULTS: In total, 46.6% (n = 7) of the cases in group III aggressive periodontitis (n = 15) and 6.66% (n = 1) in group II stage II periodontitis (n = 15) showed the presence of T. forsythia, and among them, 57.14% (n = 4) showed the presence of the glyA gene.  Conclusion: The findings of the study showed that the glyA gene may be associated with the pathogenesis of T. forsythia and could be thus a novel candidate for the future theragnostic approach to combat periodontitis.

Hemophore-like proteins of the HmuY family in the oral and gut microbiome: unraveling the mystery of their evolution.

SUMMARY: Heme (iron protoporphyrin IX, FePPIX) is the main source of iron and PPIX for host-associated pathogenic bacteria, including members of the Bacteroidota (formerly Bacteroidetes) phylum. Porphyromonas gingivalis, a keystone oral pathogen, uses a unique heme uptake (Hmu) system, comprising a hemophore-like protein, designated as the first member of the novel HmuY family. Compared to classical, secreted hemophores utilized by Gram-negative bacteria or near-iron transporter domain-based hemophores utilized by Gram-positive bacteria, the HmuY family comprises structurally similar proteins that have undergone diversification during evolution. The best characterized are P. gingivalis HmuY and its homologs from Tannerella forsythia (Tfo), Prevotella intermedia (PinO and PinA), Bacteroides vulgatus (Bvu), and Bacteroides fragilis (BfrA, BfrB, and BfrC). In contrast to the two histidine residues coordinating heme iron in P. gingivalis HmuY, Tfo, PinO, PinA, Bvu, and BfrA preferentially use two methionine residues. Interestingly, BfrB, despite conserved methionine residue, binds the PPIX ring without iron coordination. BfrC binds neither heme nor PPIX in keeping with the lack of conserved histidine or methionine residues used by other members of the HmuY family. HmuY competes for heme binding and heme sequestration from host hemoproteins with other members of the HmuY family to increase P. gingivalis competitiveness. The participation of HmuY in the host immune response confirms its relevance in relation to the survival of P. gingivalis and its ability to induce dysbiosis not only in the oral microbiome but also in the gut microbiome or other host niches, leading to local injuries and involvement in comorbidities.

Screening and characterization of aptamers recognizing the periodontal pathogen Tannerella forsythia.

Periodontal disease is one of the most common forms of inflammation. It is currently diagnosed by observing symptoms such as gingival bleeding and attachment loss. However, the detection of biomarkers that precede such symptoms would allow earlier diagnosis and prevention. Aptamers are short oligonucleotides or peptides that fold into three-dimensional conformations conferring the ability to bind molecular targets with high affinity and specificity. Here we report the selection of aptamers that bind specifically to the bacterium Tannerella forsythia, a pathogen frequently associated with periodontal disease. Two aptamers with the highest affinity were examined in more detail, revealing that their binding is probably dependent on mirolysin, a surface-associated protease secreted by the T. forsythia type-9 secretion system. The aptamers showed minimal cross-reactivity to other periodontopathogens and are therefore promising leads for the development of new tools to study the composition of the periodontitis-associated dysbiotic bacteriome as well as inexpensive new diagnostic assays.

Tannerella forsythia scavenges Fusobacterium nucleatum secreted NOD2 stimulatory molecules to dampen oral epithelial cell inflammatory response.

The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc- and MurNAc-linked peptidoglycan fragments (muropeptides) secreted by co-habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc-l-Ala-d-isoGln (MDP, muramyl dipeptide) and d-γ-glutamyl-meso-DAP (iE-DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)-like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia-F. nucleatum co-culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD-mediated inflammation. The results showed that NOD-stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia-F. nucleatum co-cultures, the NOD-stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2-stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co-habiting bacteria to dampen NOD-mediated host responses. This proof-of-principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation.

Tannerella forsythia, Fusobacterium nucleatum, and Suspected Actinomyces Causing Massive Empyema: A Case Report.

This report presents the case of a polymicrobial empyema due to Fusobacterium nucleatum, Tannerella forsythia, and suspected Actinomyces spp., presenting as several weeks of progressive shortness of breath and malaise. The patient had many risk factors for a lower respiratory tract infection, including chronic alcohol abuse, a flu-like illness months prior, and a recent invasive dental procedure. An admission CT scan showed a large right pleural effusion. Blood cultures were negative, but an aspirate from the pleural effusion showed abundant gram-positive rods that did not grow aerobically. Subsequent anaerobic cultures at a reference laboratory grew Tannerella forsythia and Fusobacterium nucleatum. This report will review the diagnostic difficulties associated with anaerobic causes of empyema in general and the specific organisms implicated in this case.

Lung Abscess Caused by Tannerella forsythia Infection: A Case Report.

Background: Tannerella forsythia is a gram-negative anaerobic bacterium commonly found in the oral cavity. It is among the common pathogenic bacteria associated with gingivitis, chronic periodontitis, and aggressive periodontitis. However, there is currently no literature discussing lung abscesses primarily caused by T. forsythia infection. Presentation: This article presents the case of a 55-year-old male with a massive lung abscess. The patient underwent ultrasound-guided percutaneous drainage, and the sample was sent for pathogen metagenomic next-generation sequencing (mNGS) testing. The test indicated that the lung abscess was primarily caused by T. forsythia infection. A literature review was conducted to understand the characteristics of this pathogen as well as its clinical features and suitable treatment approaches. Conclusion: Currently, there is no literature specifically mentioning T. forsythia as a primary pathogen causing lung abscesses. This anaerobic bacterium is commonly found in the oral cavity and is difficult to cultivate using routine culture methods. mNGS emerges as a value diagnostic method for identifying this pathogen. Treatment recommendations include drainage and antibiotic selection encompassing common periodontal pathogens such as red complex bacteria and Actinomyces.

Proteome and immune responses of extracellular vesicles derived from macrophages infected with the periodontal pathogen Tannerella forsythia.

Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia-infected macrophages were divided into two distinct vesicles, macrophage-derived EVs and T. forsythia-derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage-derived EVs were enriched with pro-inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia-derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia-derived OMVs induced pro-inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage-derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen.

Molecular docking analysis of Aza compounds with the heme-binding protein from Tannerella Forsythia.

The high biological activity and interesting optical properties of the aza compounds is known. Therefore, it is of interest to document the molecular docking analysis data of Aza compounds with the heme-binding protein from an anaerobic, Gram-negative bacterium Tannerella Forsythia. Hence, we report the optimal binding features of Aza compounds with the heme-binding protein from Tannerella Forsythia for further consideration in drug discovery against the pathogen.

Unexpected lower level of oral periodontal pathogens in patients with high numbers of systemic diseases.

Background: Periodontal disease is associated with systemic conditions such as diabetes, arthritis, and cardiovascular disease, all diseases with large inflammatory components. Some, but not all, reports show periopathogens Porphyromonas gingivialis and Tannerella forsythia at higher levels orally in people with one of these chronic diseases and in people with more severe cases. These oral pathogens are thought to be positively associated with systemic inflammatory diseases through induction of oral inflammation that works to distort systemic inflammation or by directly inducing inflammation at distal sites in the body. This study aimed to determine if, among patients with severe periodontal disease, those with multi-morbidity (or many chronic diseases) showed higher levels of periodontal pathogens. Methods: A total of 201 adult subjects, including 84 with severe periodontal disease were recruited between 1/2017 and 6/2019 at a city dental clinic. Electronic charts supplied self-reported diseases and conditions which informed a morbidity index based on the number of chronic diseases and conditions present. Salivary composition was determined by 16S rRNA gene sequencing. Results: As expected, patients with severe periodontal disease showed higher levels of periodontal pathogens in their saliva. Also, those with severe periodontal disease showed higher levels of multiple chronic diseases (multimorbidity). An examination of the 84 patients with severe periodontal disease revealed some subjects despite being of advanced age were free or nearly free of systemic disease. Surprisingly, the salivary microbiota of the least healthy of these 84 subjects, defined here as those with maximal multimorbidity, showed significantly lower relative numbers of periodontal pathogens, including Porphyromonas gingivalis and Tannerella Forsythia, after controlling for active caries, tobacco usage, age, and gender. Analysis of a control group with none to moderate periodontal disease revealed no association of multimorbidity or numbers of medications used and specific oral bacteria, indicating the importance of severe periodontal disease as a variable of interest. Conclusion: The hypothesis that periodontal disease patients with higher levels of multimorbidity would have higher levels of oral periodontal pathogens is false. Multimorbidity is associated with a reduced relative number of periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia.

Comparative Evaluation of the Inhibitory Effect of Lactobacillus rhamnosus on Halitosis-Causing Bacteria: An Invitro Microbiological Study.

AIM:  To determine the effectiveness of Lactobacillus rhamnosus in inhibiting halitosis-causing bacteria relative to other possible inhibitors, such as mouthwashes. MATERIALS AND METHODS:  This in vitro study was done using a diffusion test with three groups with 11 samples in each group: group A, Porphyromonas gingivalis; group B, Tannerella forsythia; and group C, Prevotella intermedia. At 24, 48, and 72 hours, the inhibitory effect of L. rhamnosus was tested. RESULTS: A statistically significant difference was seen for halo formation in group A, where all 11 samples showed an inhibitory effect after 72 hours. After 48 hours, seven of the 11 samples in group B and nine of the 11 samples in group C showed inhibitory effects. CONCLUSION:  The study found that L. rhamnosus had an inhibitory effect on halitosis-causing bacteria like P. gingivalis after 72 hours, which was statistically significant. The same was true for T. forsythia and P. intermedia after 48 hours. This means that L. rhamnosus has an inhibitory effect on halitosis-causing bacteria like P. gingivalis.

Characteristics of the Salivary Microbiota in Periodontal Diseases and Potential Roles of Individual Bacterial Species To Predict the Severity of Periodontal Disease.

The purposes of this study were to examine the compositional changes in the salivary microbiota according to the severity of periodontal disease and to verify whether the distribution of specific bacterial species in saliva can distinguish the severity of disease. Saliva samples were collected from 8 periodontally healthy controls, 16 patients with gingivitis, 19 patients with moderate periodontitis, and 29 patients with severe periodontitis. The V3 and V4 regions of the 16S rRNA gene in the samples were sequenced, and the levels of 9 bacterial species showing significant differences among the groups by sequencing analysis were identified using quantitative real-time PCR (qPCR). The predictive performance of each bacterial species in distinguishing the severity of disease was evaluated using a receiver operating characteristic curve. Twenty-nine species, including Porphyromonas gingivalis, increased as the severity of disease increased, whereas 6 species, including Rothia denticola, decreased. The relative abundances of P. gingivalis, Tannerella forsythia, Filifactor alocis, and Prevotella intermedia determined by qPCR were significantly different among the groups. The three bacterial species P. gingivalis, T. forsythia, and F. alocis were positively correlated with the sum of the full-mouth probing depth and were moderately accurate at distinguishing the severity of periodontal disease. In conclusion, the salivary microbiota showed gradual compositional changes according to the severity of periodontitis, and the levels of P. gingivalis, T. forsythia, and F. alocis in mouth rinse saliva had the ability to distinguish the severity of periodontal disease. IMPORTANCE Periodontal disease is one of the most widespread medical conditions and the leading cause of tooth loss, imposing high economic costs and an increasing burden worldwide as life expectancy increases. Changes in the subgingival bacterial community during the progression of periodontal disease can affect the entire oral ecosystem, and bacteria in saliva can reflect the degree of bacterial imbalance in the oral cavity. This study explored whether the specific bacterial species in saliva can distinguish the severity of periodontal disease by analyzing the salivary microbiota and suggested P. gingivalis, T. forsythia, and F. alocis as biomarkers for distinguishing the severity of periodontal disease in saliva.

Hemophore-like proteins produced by periodontopathogens are recognized by the host immune system and react differentially with IgG antibodies.

Aims: Hemophore-like proteins sequester heme from host hemoproteins. We aimed to determine whether the host immune system can recognize not only Porphyromonas gingivalis HmuY but also its homologs expressed by other periodontopathogens, and how periodontitis influences the production of respective antibodies. Methods: The reactivity of total bacterial antigens and purified proteins with serum IgG antibodies of 18 individuals with periodontitis and 17 individuals without periodontitis was examined by enzyme-linked immunosorbent assay (ELISA). To compare IgG reactivity between groups with and without periodontitis and within the various dilutions of sera, statistical analysis was performed using the Mann-Whitney U-test and two-way ANOVA test with the post-hoc Bonferroni test. Results: Individuals with periodontitis produced IgG antibodies reacting more strongly not only with total P. gingivalis antigens (P = 0.0002; 1:400) and P. gingivalis HmuY (P = 0.0016; 1:100) but also with Prevotella intermedia PinA (P = 0.0059; 1:100), and with low efficiency with P. intermedia PinO (P = 0.0021; 1:100). No increase in the reactivity of IgG antibodies with Tannerella forsythia Tfo and P. gingivalis HusA was found in individuals with periodontitis. Conclusions: Although hemophore-like proteins are structurally related, they are differentially recognized by the host immune system. Our findings point to specific antigens, mainly P. gingivalis HmuY and P. intermedia PinA, whose immunoreactivity could be further investigated to develop markers of periodontitis.

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.

Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR).

The quantitative polymerase chain reaction (qPCR) is a variant of PCR aimed to detect and quantify a targeted DNA molecule. This is made through the addition of probes labeled with fluorescent molecules that emit fluorescence within each amplification cycle, resulting in fluorescence values proportional to the amount of accumulated PCR product. This chapter presents the detailed procedures for quantification of different periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Streptococcus oralis, and Fusobacterium spp.) using qPCR. It also includes the description of the most frequent problems encountered, how to solve them, and recommendations to minimize the risks for laboratory staff handling oral samples. In addition, a detailed protocol for multiplex qPCR to detect and quantify Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia is also included.