Filifactor Alocis Research Articles

Characterization of thioredoxin-thioredoxin reductase system in Filifactor alocis.

Filifactor alocis is a newly appreciated member of the periodontal community with a strong periodontal disease correlation. Little is known about the survival mechanisms by which F.alocis copes with oxidative stress and establishes the infection within the local inflammatory microenvironment of the periodontal pocket. The aim of this study is to investigate if F.alocis putative peroxiredoxin/AhpC protein FA768 may constitute an alkyl hydroperoxide reductase system utilizing putative thioredoxin reductase protein FA608, and putative thioredoxin/glutaredoxin homolog FA1411/FA455. FA768, FA608, FA1411 and FA455 proteins from F.alocis were expressed and purified from Escherichia coli. Insulin and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reduction assays were performed to determine if purified FA1411 and FA455 proteins could be a substrate for FA608. The peroxidase activity of FA768 was examined by measuring its ability to reduce hydrogen peroxide (H2O2) with FA608 and FA1411/FA455 provided as the reducing systems. Further, the hydroperoxide substrate specificity of FA768 was analyzed by monitoring the NADPH oxidation in the presence of different peroxides, including H2O2, cumyl hydroperoxide (CHP), and tert-butyl hydroperoxide (t-BHP). In this study, we have demonstrated the existence of a functioning thioredoxin-dependent alkyl hydroperoxide system in F.alocis. This system is comprised of a thioredoxin reductase (FA608), a thioredoxin/glutaredoxin homolog (FA1411/FA455), and a typical 2-cysteine peroxiredoxin/AhpC (FA768). FA608, together with FA1411/FA455, can function as a thioredoxin reductase system to reduce insulin, DTNB, and FA768. FA455 is a glutaredoxin-like protein with thioredoxin functions in F.alocis. Both the FA768/FA608/FA1411 and FA768/FA608/FA455 reductase systems were NADPH-dependent and exhibited specificity for broad hydroperoxide substrates H2O2, CHP, and t-BHP. This is the first study of a thioredoxin dependent alkyl hydroperoxide system from a periodontal pathogen. This system is proposed to protect F.alocis against oxidative stress due to the likely absence of a catalase or an additional peroxiredoxin homolog.

Microbiota of Peri-Implant Healthy Tissues, Peri-Implant Mucositis, and Peri-Implantitis: A Comprehensive Review.

Understanding the microbiological profiles of peri-implant conditions is crucial for developing effective preventive and therapeutic strategies. This narrative review analyzes the microbial profiles associated with healthy peri-implant sites, peri-implant mucositis, and peri-implantitis, along with related microbiological sampling and analyses. Healthy peri-implant sites are predominantly colonized by Streptococcus, Rothia, Neisseria, and Corynebacterium species, in addition to Gram-positive cocci and facultatively anaerobic rods, forming a stable community that prevents pathogenic colonization and maintains microbial balance. In contrast, peri-implant mucositis shows increased microbial diversity, including both health-associated and pathogenic bacteria such as red and orange complex bacteria, contributing to early tissue inflammation. Peri-implantitis is characterized by even greater microbial diversity and a complex pathogenic biofilm. Predominant pathogens include Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, and unique species like Filifactor alocis and Fretibacterium fastidiosum. Additionally, less common species such as Staphylococcus and Enterobacteriaceae, contributing to disease progression through biofilm formation and increased inflammatory response, along with EBV and human cytomegalovirus with a still not defined role, and Candida albicans contribute to disease progression through biofilm formation, immune modulation, and synergistic inter-kingdom interactions. Future research should standardize diagnostic criteria, employ advanced molecular techniques, integrate microbial data with clinical factors, and highlight inter-kingdom interactions.

The impact of Filifactor alocis on the severity of periodontitis among diabetic and non-diabetic patients: a narrative review

The impact of Filifactor alocis on the severity of periodontitis among diabetic and non-diabetic patients: a narrative review

Periodontal inflammatory and microbial profiles in healthy young African Americans and Caucasians.

This study aimed to compare microbial and inflammatory profiles in periodontally/systemically healthy African American (AA) and Caucasian (C) individuals. Thirty-seven C and 46 AA aged from 5 to 25 years were evaluated regarding periodontal disease, caries, microbial subgingival profile via 16-s sequencing, as well as salivary and gingival crevicular fluid (GCF) inflammatory profile via multiplex assay. Greater probing depth percentage was detected in AA (p = .0075), while a higher percentage of caries index (p = .0069) and decayed, missing, filled teeth (DMFT) index (p = .0089) was observed in C, after adjusting for number of teeth, sex and age. Salivary levels of IL-6, IL-8 and TNFα were higher for C, whereas GCF levels of eotaxin, IL-12p40, IL-12p70, IL-2 and MIP-1α were higher in AA (p < .05). Different microbial profiles were observed between the races (p = .02). AA presented higher abundance of periodontopathogens (such as Tanerella forsythia, Treponema denticola, Filifactor alocis, among others), and C presented more caries-associated bacteria (such as Streptococcus mutans and Prevotella species). Bacillaceae and Lactobacillus species were associated with higher DMFT index, whereas Fusobacterium and Tanerella species with periodontal disease parameters. A different inflammatory and bacterial profile was observed between healthy AA and C, which may predispose these races to higher susceptibility to specific oral diseases.

Subgingival microbiome in periodontal disease and comorbid pathology (meta-analysis)

The problem of chronic periodontitis (CP) is actively discussed due to the recognition of the fact that periodontal microbial damage is closely related to a number of systemic diseases and probably plays an important role in the occurrence of comorbid pathology. The aim of the meta-analysis was to characterize the composition of the subgingival microbiome and to determine the peculiarities of the formation of associations of the new periodontal pathogen Filifactor alocis with other I and II order periodontal pathogenic bacteria, as well as with the commensal bacteria colonizing this biotope. The study presents data of patient examination with obligatory use of polymerase chain reaction methods and sequencing of 16S rRNA genes in 1529 healthy individuals and 2394 patients with CP, 136 patients with CP and concomitant atherosclerosis, 258 patients with CP and concomitant type 2 diabetes mellitus. It was confirmed that the basis of the oral microbiome under normal circumstances is composed of representatives of microaerophilic streptococci, corynebacteria, lactobacilli, as well as representatives of the Veillonella and Sphingobacterium genera. 16S sequencing and bioinformatic analysis allowed us to specify the taxonomic place of the new pathogen F. alocis, as well as representatives of normal microbiota in CP and comorbid somatic pathology.

Association of Filifactor alocis and its RTX toxin gene ftxA with periodontal attachment loss, and in synergy with Aggregatibacter actinomycetemcomitans.

The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

Enhancement of innate immunity in gingival epithelial cells by vitamin D and HDAC inhibitors.

The human host defense peptide LL-37 is a component of the innate immune defense mechanisms of the oral cavity against colonization by microbes associated with periodontal disease. We have previously shown that the active form of vitamin D, 1,25(OH)2D3, can induce the expression of LL-37 in gingival epithelial cells (GEC), and prevent the invasion and growth of periopathogenic bacteria in these cells. Further, experimental vitamin D deficiency resulted in increased gingival inflammation and alveolar bone loss. Epidemiological studies have shown associations between vitamin D deficiency and periodontal disease in humans, suggesting application of vitamin D could be a useful therapeutic approach. Further, since we have shown the local activation of vitamin D by enzymes expressed in the GEC, we hypothesized that we could observe this enhancement with the stable, and inexpensive inactive form of vitamin D, which could be further increased with epigenetic regulators. We treated 3-dimensional primary cultures of GEC topically with the inactive form of vitamin D, in the presence and absence of selected histone deacetylase (HDAC) inhibitors. LL-37 mRNA levels were quantified by quantitative RT-PCR, and inhibition of invasion of bacteria was measured by fluorescence microscopy. Vitamin D treatment led to an induction of LL-37 mRNA levels, as well as an inhibition of pro-inflammatory cytokine secretion. This effect was further enhanced by HDAC inhibitors, most strongly when the HDAC inhibitor, phenyl butyrate (PBA) was combined with Vitamin D3. This was observed both in solution and in a prototype gel formulation using sodium butyrate. Finally, this combination treatment led to an increase in the antimicrobial activity against infection by Porphyromonas gingivalis and Filifactor alocis, bacteria associated with periodontal lesions, as well as herpes simplex virus, which has also been shown to be associated with periodontal lesions. Our results demonstrate that a combination of inactive vitamin D and sodium butyrate could be developed as a safe treatment for periodontal disease.

Evaluation of microbiome in primary and permanent dentition in grade C periodontitis in young individuals.

The aim of the present study was to evaluate the subgingival microbiome in patients with grade C molar-incisor pattern periodontitis (C-MIP) affecting the primary or permanent dentitions. DNA was isolated from subgingival biofilm samples from diseased and healthy sites from 45 C-MIP patients and subjected to phylogenetic microarray analysis. C-MIP sites were compared between children affected in the primary to those affected in the permanent dentitions. Within-subject differences between C-MIP-affected sites and dentition-matched healthy sites were also evaluated. C-MIP sites of subjects affected in the primary dentition showed partially overlapping but distinct microbial communities from C-MIP permanent dentition sites (p<0.05). Differences were due to increased levels in primary C-MIP sites of certain species of the genera Capnocytophaga and Leptotrichia, while C-MIP permanent dentition sites showed higher prevalence of Filifactor alocis. Aggregatibacter actinomycetemcomitans (Aa) was among species seen in high prevalence and levels in both primary and permanent C-MIP sites. Moreover, both permanent and primary C-MIP sites showed distinct microbial communities when compared to dentition-matched healthy sites in the same subject (p<0.01). Primary and permanent teeth with C-MIP showed a dysbiotic microbiome, with children affected in the primary dentition showing a distinct profile from those affected in the permanent dentition. However, Aa was enriched in both primary and permanent diseased sites, confirming that this microorganism is implicated in C-MIP in both dentitions.

Glucose-6-Phosphatase-Dehydrogenase activity as modulative association between Parkinson's disease and periodontitis.

https://www.bfarm.de/DE/Das-BfArM/Aufgaben/Deutsches-Register-Klinischer-Studien/_node.html, identifier DRKS00005388.

Bacteria of genus &lt;i&gt;Filifactor&lt;/i&gt; in patients with periodontitis and type 2 diabetes in accordance with metagenomic analysis of the periodontal microbiome

Introduction. Periodontal diseases are a common pathology with chronic periodontitis (CP) being the most severe form. This polymicrobial disease has become a problem of great importance in recent years due to the possibility of development of systemic effects associated with this condition. CP is often combined with type 2 diabetes (T2D). The main cause of the occurrence and development of periodontal pathology is played by the bacteria Filifactor alocis, the least studied and most recently discovered periodontal pathogen.&#x0D; The objective of this study was to identify bacteria of genus Filifactor as part of the periodontal microbiome associated with CP and T2D and to clarify the mechanisms of their possible influence on associated metabolic processes according to comparative metagenomic analysis.&#x0D; Materials and methods. A metagenomic study of the microbiome of periodontal pocket samples from 28 patients with CP associated with T2D and 22 patients with CP, as well as the microbiome of dental gingival sulcus samples from 19 clinically healthy individuals was performed. 16S-sequencing of the ribosomal RNA gene was used to determine the taxonomic composition of the microbiome. Prediction of metabolic pathways involving the microbiome was performed with the help of the shotgun method.&#x0D; Results. Filifactor bacteria were the one of the most frequent microorganisms only in patients with CP associated with T2D. The rate of identification of these bacteria was correlated with low predicted metagenomic levels of fatty acid biosynthesis and pyrimidine metabolism in the affected area.&#x0D; Conclusion. The detection frequency of Filifactor bacteria in patients associated with CP and T2D is negatively correlated with the selected features of putative metabolic pathways of the microbiome, which include fatty acid biosynthesis and pyrimidine metabolism.

Extracellular vesicles from periodontal pathogens regulate hepatic steatosis via Toll-like receptor 2 and plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is associated with nonalcoholic fatty liver disease (NAFLD) by lipid accumulation in the liver. In this study, we showed that extracellular vesicles (EVs) from the periodontal pathogens Filifactor alocis and Porphyromonas gingivalis induced steatosis by inducing PAI-1 in the liver and serum of mice fed a low-fat diet. PAI-1 induction was not observed in TLR2-/- mice. When tested using HEK-Blue hTLR2 cells, human TLR2 reporter cells, the TLR2-activating ability of serum from NAFLD patients (n=100) was significantly higher than that of serum from healthy subjects (n=100). Correlation analysis confirmed that PAI-1 levels were positively correlated with the TLR2-activating ability of serum from NAFLD patients and healthy subjects. Amphiphilic molecules in EVs were involved in PAI-1 induction. Our data demonstrate that the TLR2/PAI-1 axis is important for hepatic steatosis by EVs of periodontal pathogens.

Filifactor alocis enhances survival of Porphyromonas gingivalis W83 in response to H2 O2 -induced stress.

A dysbiotic microbial community whose members have specific/synergistic functions that are modulated by environmental conditions, can disturb homeostasis in the subgingival space leading to destructive inflammation, plays a role in the progression of periodontitis. Filifactor alocis, a gram-positive, anaerobic bacterium, is a newly recognized microbe that shows a strong correlation with periodontal disease. Our previous observations suggested F. alocis to be more resistant to oxidative stress compared to Porphyromonas gingivalis. The objective of this study is to further determine if F. alocis, because of its increased resistance to oxidative stress, can affect the survival of other 'established' periodontal pathogens under environmental stress conditions typical of the periodontal pocket. Here, we have shown that via their interaction, F. alocis protects P. gingivalis W83 under H2 O2 -induced oxidative stress conditions. Transcriptional profiling of the interaction of F. alocis and P. gingivalis in the presence of H2 O2 -induced stress revealed the modulation of several genes, including those with ABC transporter and other cellular functions. The ABC transporter operon (PG0682-PG0685) of P. gingivalis was not significant to its enhanced survival when cocultured with F. alocis under H2 O2 -induced oxidative stress. In F. alocis, one of the most highly up-regulated operons (FA0894-FA0897) is predicted to encode a putative manganese ABC transporter, which in other bacteria can play an essential role in oxidative stress protection. Collectively, the results may indicate that F. alocis could likely stabilize the microbial community in the inflammatory microenvironment of the periodontal pocket by reducing the oxidative environment. This strategy could be vital to the survival of other pathogens, such as P. gingivalis, and its ability to adapt and persist in the periodontal pocket.

Regenerative endodontic procedures in immature permanent teeth with pulp necrosis: the impact of microbiology on the clinical and radiographic outcome

AimsThe current study aimed to determine how the regenerative endodontic procedures (REP) disinfection strategy influences overall bacterial load and REP outcomes. Different bacterial species in the teeth were also examined.MethodsA previously reported non-randomized controlled clinical research on REP ± leukocyte and platelet-rich fibrin gathered microbial samples from 14 of 29 patients during REP (LPRF). Four microbiological samples were obtained in two treatment sessions. S1 and S2 were taken before and after the first irrigation with 1.5% NaOCl and saline. Samples S3 and S4 were obtained before and after rinsing with 17% EDTA in the second treatment session. Microbial samples were identified using quantitative polymerase chain reaction with species-specific primers.ResultsThe total bacterial load recovered from patients showed a significant (p &amp;lt; 0.05) decrease after the first treatment and was maintained throughout the second treatment. Fusobacterium nucleatum, Treponema denticola, and Enterococcus faecalis were the most prevalent species in root canals, detected in all analyzed cases (100%), followed by Prevotella intermedia and Tannerella forsythia, both in six of 14 (42.9%) cases. The presence of these abundant species was significantly reduced after S1. Parvimonas micra was present in four of 14 (28.6%) cases and Actinomyces naeslundii in two of 14 (14.3%) cases. Filifactor alocis, Porphyromonas endodontalis, and Porphyromonas gingivalis were each detected in only one of 14 (7.1%) cases. No statistical correlation could be made between bacterial species and clinical or radiographic outcomes due to the small sample size. In the LPRF group, two cases required retreatment due to early post-treatment flare-up, and two other cases presented radiographically a persistent apical periodontitis 3 years after treatment. In the control group, all analyzed cases were clinically asymptomatic after treatment, and radiographically the final periapical index score at the last recall revealed healthy periapices.ConclusionThe REP disinfection protocol of the present study seems to be satisfactorily effective in reducing the total bacterial load, omitting clinical symptoms, and inducing periapical bone healing in immature permanent teeth with pulp necrosis. LPRF seems to prevent these outcomes from being achieved and should consequently not be recommended in REP.

AETIOLOGICAL CHARACTERISTICS OF DENTAL IMPLANT FAILURE

Dental implant placement is a predictable and widely used procedure in modern dentistry. Nevertheless, several factors can negatively affect the functioning and duration of implant restoration. Considering the significant data supporting the role of the microbial factor in the development of tissue inflammation around the prosthesis, the purpose of this review was to investigate the etiological structure of implant loss from a microbiological point of view. Materials and methods. We conducted a comprehensive content analysis of scientific publications available in the PubMed/MEDLINE and Google Scholar databases. Results and discussion. Six groups of microorganisms are known as potential contributor to the dental biofilm. These groups are designated by a specific color – yellow, green, purple, orange and red. The orange and red groups include pathogens of periodontal inflammation, and also play a significant role in the development of peri-implant (PI) inflammation. Implants and teeth share histopathological and ecological similarities, thus, we can suggest that the microbial communities around these structures are similar. In the studies, the composition of the PI-associated complex of microorganisms including Porphyromonas gingivalis, Porphyromonas endodontalis, Tannerella forsythia, Filifactor alocis, Fretibacterium fastidiosum, Desulfobulbus spp. and Treponema lecithinolyticum was decsribed. It has been reported that the PI microbiome is specific to the site where an implant is placed, and the microbial composition of the biofilm of the contralateral healthy sites is more similar to the spectrum of healthy prostheses, including other subjects, than those from the same oral cavity. We can suggest that the changes in the implant microbiocenosis are dynamic. Several studies have shown the long-term consequences of dental implant restoration that can include the modification of the microbiome consisting in the progressive increase of P. gingivalis, T. forsythia, A. actinomycetemcomitans and Prevotella intermedia within 3-6 months following the procedure of implant placement. There is also an opinion about the peculiarities of the biofilm microbiota of the peri-implant tissues in case of early and late loss of the dental prosthesis. Bacteria are an important component of the microbiocenosis in any ecological niche, but the role of archaea is often overlooked. Methanogenic archaea, and Methanobrevibacter oralis are components of the normal oral microbiome, but their considerable prevalence may also be associated with peri-implant tissue inflammation. Conclusions. We have elucidated the complex nature of microbial communities within peri-implant sites and established an association between peri-implant diseases and the dysbiosis of subgingival microbial communities. Our study underscores the pivotal role of microbiota in peri-implant diseases.

Quantitative investigation of the bacterial content of periodontal abscess samples by real-time PCR

ObjectivesPeriodontal abscesses, which are part of the acute periodontal disease group characterized by the destruction of periodontal tissue with deep periodontal pockets, bleeding on probing, suppuration, and localized pus accumulation, cause rapid destruction of tooth-supporting tissues. This study aimed to evaluate the microbial content of periodontal abscesses by specific and culture-independent qPCR. MethodsThis study was conducted on 30 volunteers diagnosed with periodontal abscesses and presenting with complaints of localized pain, swelling, and tenderness in the gingiva. Genomic DNA was isolated from the samples taken. Escherichia coli bacteria were used for the standard curve created to calculate the prevalence of target bacteria in the total bacterial load. 16S rRNA Universal primers were used to assess the total bacterial load and prevalence. Bacterial counts were analyzed with Spearman's rank correlation coefficients (ρ) matrix. ResultsFrom the analysis of Real-Time PCR, Porphyromonas gingivalis (30, 100%), Prevotella intermedia (30, 100%), and Fusobacterium nucleatum (30, 100%) were detected in all samples. Campylobacter rectus (29, 96.6%), Porphyromonas endodontalis (29, 96.6%), Tannerella forsythia (28, 93.3%), Filifactor alocis (28, 93.3%), and Actinomyces naeslundii (28, 93.3%) were also frequently detected. ConclusionsPeriodontal abscesses were found to be polymicrobial, and not only periodontal pathogens appeared to be associated with the development of periodontal abscesses. The presence, prevalence, and number of Porphyromonas endodontalis and Propionibacterium acnes in the contents of periodontal abscesses were determined for the first time in our study. Further studies are needed to better understand the roles of bacteria in periodontal disease, including abscesses.

Induction of TNF-α by Filifactor alocis in THP-1 macrophagic cells

ObjectivesFilifactor alocis is an emerging periodontal pathogen, and macrophage-produced tumor necrosis factor-α (TNF-α) plays important roles in periodontal pathogenesis. In this study, we investigated F. alocis-stimulated TNF-α production in THP-1 macrophagic cells. DesignPhorbol 12-myristate 13-acetate-differentiated THP-1 macrophagic cells were challenged with F. alocis ATCC 35896 for various durations. TNF-α mRNA expression and protein secretion were determined using RT-PCR and ELISA, respectively. Activation of protein kinases and transcription factor proteins was evaluated by Western blot analysis. ResultsLive F. alocis stimulated THP-1 cells to produce TNF-α in a dose-dependent manner. However, glutaraldehyde-killed or heat-killed F. alocis showed no effectiveness for TNF-α induction. In contrast, both live and killed Porphyromonas gingivalis robustly increased TNF-α expression. Furthermore, F. alocis was unable to stimulate TNF-α expression in Toll-like receptor 2 (TLR2) knockout THP-1 cells. F. alocis activated all three mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Pharmacological inhibition of ERK and JNK, but not p38, significantly reduced F. alocis-induced TNF-α production. Finally, increased levels of phospho-c-Jun were detected in F. alocis-stimulated THP-1 cells. ConclusionsThese results suggest that F. alocis induces TNF-α production in THP-1 macrophagic cells primarily by activating the TLR2, JNK, and c-Jun pathways.

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.

Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis

ObjectiveThis study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. MethodsThe standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Resultsobtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%−72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). ConclusionsThe uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.

Environmental influences on Streptococcus sanguinis membrane vesicle biogenesis.

Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell communication, information is scarce about their general biology in the context of community members and the environment. Streptococcus sanguinis, a Gram-positive oral commensal, is prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1β, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. Our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors and plays an important role in communication with host cells.

Whole Genome Sequencing and Phenotypic Analysis of Antibiotic Resistance in Filifactor alocis Isolates.

There is scarce knowledge regarding the antimicrobial resistance profile of F. alocis. Therefore, the objective of this research was to assess antimicrobial resistance in recently obtained F. alocis clinical isolates and to identify the presence of antimicrobial resistance genes. Isolates were obtained from patients with periodontal or peri-implant diseases and confirmed by sequencing their 16S rRNA gene. Confirmed isolates had their genome sequenced by whole genome sequencing and their phenotypical resistance to nine antibiotics (amoxicillin clavulanate, amoxicillin, azithromycin, clindamycin, ciprofloxacin, doxycycline, minocycline, metronidazole, and tetracycline) tested by E-test strips. Antimicrobial resistance genes were detected in six of the eight isolates analyzed, of which five carried tet(32) and one erm(B). Overall, susceptibility to the nine antibiotics tested was high except for azithromycin in the isolate that carried erm(B). Moreover, susceptibility to tetracycline, doxycycline, and minocycline was lower in those isolates that carried tet(32). The genetic surroundings of the detected genes suggested their inclusion in mobile genetic elements that might be transferrable to other bacteria. These findings suggest that, despite showing high susceptibility to several antibiotics, F. alocis might obtain new antimicrobial resistance traits due to its acceptance of mobile genetic elements with antibiotic resistance genes in their genome.