Diagnostic value of microbiome biomarkers of the periodonite microbiome in patients with the association of chronic periodontitis and diabetes mellitus type 2.

The place of high-tech methods of molecular biology in clinical laboratory diagnostics of various diseases and the development of a system of biomarkers as an important component of diagnostic research is currently attracting the closest attention of the scientific community. In this paper, an attempt is made to use high-tech metagenomic analysis to solve problems that arise due to the high frequency of association of periodontal diseases with systemic pathology, in particular, with type 2 diabetes mellitus. The aim of the study was to determine the taxonomic and metabolic features of the microbiome of periodontal tissues in periodontal diseases associated with type 2 diabetes mellitus, as a model of the ratio of local and systemic effects of periodontal pathogenic bacteria. The study included 16S shotgun sequencing of bacterial DNA as part of biological material from periodontal pockets/dentoalveolar furrows of 46 people - 15 patients with chronic periodontitis associated with type 2 diabetes mellitus, 15 patients with chronic periodontitis unrelated to systemic pathology, as well as 16 healthy people in the control group, followed by bioinformatic processing of the data obtained. The obtained data allowed us to establish the taxonomic features of the periodontal microbiome in the association of chronic periodontitis with type 2 diabetes mellitus, which included the predominance of representatives of the families Prevotellaceae and Spirochaetaceae in its composition. The features of metabolic processes in periodontal tissues with the participation of the microbiome were also revealed, which consisted in an increase in the exchange of cysteine and methionine against the background of a decrease in the metabolism of pyrimidine, methane, sphingolipids, and the synthesis of fatty acids, which are of diagnostic value in assessing the condition of patients with type 2 diabetes mellitus.

[Filifactor alocis and its role in the etiology of chronic periodontitis]. / Filifactor alocis i ego rol' v etiologii khronicheskogo parodontita.

The review is devoted to the analysis of modern ideas about the role of bacteria Filifactor alocis in the etiology of chronic periodontitis. The study of these bacteria, discovered in 1985, is complicated by the difficulty of their detection with cultural methods. According to modern researches, the bacteria F.alocis with good reason can be included in the red complex of periodontal pathogens as the most important pathogens of chronic periodontitis. F.alocis is a synergist of such a key pathogen Porphyromonas gingivalis, as well as a frequent satellite of Fusobacterium nucleatum and, somewhat less frequently, Aggregatibacter actinomycetemcomitans. F.alocis is practically not found in healthy people (except for smokers), with a high frequency accompanies the aggressive course of periodontal disease, and also recorded in endodontitis. Due to the ability to participate in the metabolism of arginine, expressed protease activity, a wide range of virulence factors, F.alocis not only colonizes the periodontal tissues, but also significantly affects the formation of the community of periodontal microorganisms (including viruses), contributing to their invasion of epithelial tissues. F. alocis has a number of unique properties, including resistance to oxidative stress conditions in the home defeat, induction of apoptosis of epithelial cells, extracellular matrix degradation of periodontal tissues, activation of proinflammatory cytokines formulation in sites of its presence, suppression of protective reactions of neutrophilic granulocytes, inhibition of the process of complement activation.

"Shielding" of Cytokine Induction by the Periodontal Microbiome in Patients with Periodontitis Associated with Type 2 Diabetes Mellitus.

Periodontal diseases, especially those with polymicrobial etiology, are often associated with type 2 diabetes mellitus, proceeding more severely and affecting the course of diabetes mellitus. Recently, this feature has been associated with the ability of periodontopathogen microflora to cause not only a local infectious process in the oral cavity, but also to interact with the human immune system and induce various systemic effects. We investigated changes in the salivary cytokine profile of patients with chronic periodontitis, associated and not associated with type 2 diabetes mellitus. We observed a statistically significant decrease of MCP-1/CCL2, GM-CSF, IL-5, IL-6, and IFN-γ in the saliva of patients with chronic periodontitis associated with type 2 diabetes mellitus in comparison with patients with chronic periodontitis only. All of these cytokines are associated with macrophage activation. These data are an important contribution to the elucidation of the mechanism of periodontopathogens involvement in the manifestation of the systemic effects of type 2 diabetes.

[Control of primary adhesion of microorganisms and formation of biofilms on stomatological materials used for transdental implantation in dental stabilizing operations.]

Transdental implantation is an effective way to restore the lost biomechanical properties of a resected or amputated tooth. The choice of structural materials for the implant is the most important stage of treatment, in relation to the adhesion of aggressive microbiota to them. To characterize the adhesion of microorganisms of periodontopathogenic and cariogenic groups in vitro to experimental samples of zirconium dioxide and titanium alloys using cultural and electronic microscopic methods of adhesion evaluation as the first stage of biofilm formation. Samples for the experiment were prepared in the form of a tablet of standard form, on which the test strains were applied in an amount of 106CFU/ml. After shaking the unattached cells with ultrasound, they were seeded into dense nutrient medium to determine their number. In total, 14 strains of perodontopathogenic and cariogenic groups (including 3 reference strains) were used in the experiment. Scanning electron microscopy was used to visualize the adhesion of microorganisms. The results of the adhesion test to titanium nickelide and zirconium dioxide showed a significant reduction in adhesion for all microorganism species. In all variants (with all strains) the adhesion values to titanium nickelide and zirconium dioxide were statistically significantly lower than when using samples from a traditional titanium alloy. In scanning electron microscopy, single cells of test strains of perodontopathogenic microorganisms were determined on zirconium dioxide samples, while a considerable number of cells and the initial phase of biofilm formation were observed on the compared structural materials. Zirconium dioxide can be considered as an optimal choice material for the manufacture of transdental implants, which, in terms of its technological characteristics and low adhesion characteristics of microbes, is superior to the traditionally used titanium alloys.

Metagenomic Analysis of Gingival Sulcus Microbiota and Pathogenesis of Periodontitis Associated with Type 2 Diabetes Mellitus.

Biofilm of the gingival sulcus from 22 patients with type 2 diabetes mellitus and periodontitis, 30 patients with periodontitis not complicated by diabetes mellitus (reference group), and 22 healthy volunteers without signs of gingival disease (control group) was studied by quantitative PCR. Quantitative analysis for the content of P. gingivalis, T. forsythia, A. ctinomycetemcomitans, T. denticola, P. intermedia, F. nucleatum/periodonticum, and P. endodontalis in the dental plaque was performed with a Dentoscreen kit. The presence of other bacterial groups was verified by metagenomic sequencing of the 16S rRNA gene to evaluate some specific features of the etiological factor for periodontitis in type 2 diabetes mellitus. Specimens of the Porphiromonadaceae and Fusobacteriaceae families were characterized by an extremely high incidence in combined pathology. The amount of Sphingobacteriaceae bacteria in the biofilm was shown to decrease significantly during periodontitis. Metagenomic analysis confirmed the pathogenic role of microbiota in combined pathology, as well as the hypothesis on a possible influence of periodontitis on the course and development of type 2 diabetes mellitus.

[PREVALENCE OF GENETIC MARKERS OF RESISTANCE TO ANTIBIOTICS IN BIOFILM-FORMING STRAINS OF OBLIGATE AND ELECTIVE ANAEROBES].

AIM: Comparative study of frequency of detection of genetic markers of resistance to antibiotics forming in anaerobic bacteria under the conditions of mixed biofilms in a clinical setting and comparison of data of phenotypic and genotypic methods of study. MATERIALS AND METHODS: 66 strains of bacteria forming biofilm with PCR detection of antibiotics were studied: Streptococcus sanguinis, Streptococcus salivarius, Staphylococcus aureus, Staphylococcus epi- dermidis, Enterococcusfaecalis, Klebsiellapneumoniae, Pseudomonas aeruginosa and anaero- bic pathogens - Porphyromonasgingivalis, Tannerella forsythia, Parvinonas micra, Prevotella intermedia. Modelling of microbial biofilms in vitro and scanning electron microscopy were carried out. RESULTS: The studied strains of resident and pathogenic microbiota were established to have genes that code resistance to P-lactam antibiotics; carbapenems, macrolides, tetra- cyclines. Genetic markers of resistance to P-lactam antibiotics (STX-M 14 MECA - cepha- losporines), including carbapenems.(VIM and NDM, but not Oxa-48), glycopeptides (VanA and VanB), macrolides (ERM), tetracycline (Tet) and QNRB plasmids (fluoroquinolones) were detected in strains by PCR. CONCLUSION: The most frequently used preparations in dental practice - metronidazole and lincomycin (for the last 20 - 30 years) have shown the highest number of resistant strains - 52.3 and 22.7%, respectively. The frequency of detection of genetic markers of resistance to other studied preparations did not exceed 2.5 - 11.4%. Minimal quantity of resistant strains of anaerobic bacteria was detected for carbapenems and fluoroquinolones.

[The application of scanning electronic microscopy and bio-reactor for evaluating eradication of microbial bio-film of root channels under endodontic treatment.]

According to the results of endodontic treatment of chronic forms of odontotis and periodontitis the rate of complications continues to be high - from 32% to 50% that os related to characteristics of microbial bio-film. The studies of this object in root canals and methods of eradication of bio-films are at their initial stage. The purpose of study is to provide a laboratory substantiation and evaluation of efficacy of complex effect of ultrasound processing and photo-activating disinfection of root of tooth in case of chronic forms of odontotis and periodontitis and also evaluation of endodontic treatment using scanning electronic microscopy. The micro-flora of root canals was examined using bacteriological method (4 bacterial and 2 fungous clinical isolates). The effect of ultrasound processing in complex with the system, photo-activating disinfection on micro-flora of root canal was evaluated using a bio-reactor. The analysis of bio-films of system of root canals of tooth in patients with chronic forms of odontotis (52 patients) and periodontitis (16 patients) are implemented using scanning electronic microscopy. The established availability of abundant microbial bio-film in orifices of dentinal tubules and obturation of dentinal tubules in particular cases. The features of formation of curves of growth of population of bacteria of oral micro-flora (Porphyromonas gingivalis; Fusobacterium nucleatum; Streptococcus sanguinis; Streptococcus mutans; Candida albicans; Candida krusei) as a result of complex effect of photo-dynamic activation and ultrasound as compared with control without such. The complex effect inhibits growth of microbial populations of oral bacteria and fungi species Candida.

Clinical-microbiological research of action ozone therapy and light-emetting diode radiation of red range (630 nanometers) on microflora of the hole extracted toothatalveolitis and limited osteomyelitis of jaws. / Kliniko-mikrobiologicheskoe issledovanie deistviya ozonoterapii i svetodiodnogo izlucheniya krasnogo diapazona (630 nm) na mikrofloru lunki udalennogo zuba pri al'veolite i ogranichennom osteomielite chelyustei.

As a result of cliniko-microbiological research the data testifying to substantial improvement of efficiency of antimicrobictherape at inclusion in a complex of medical actions at alveolitis and the limited osteomyelitis of a jow ozone therapy in a combination with a light-emettinf diode irradiation of the hole extracted teeth red ( 630 nanometers) are obtained by light.

[THE MOLECULAR TECHNIQUES OF DIAGNOSTIC OF GINGIVITIS AND PERIODONTITIS IN HIV-INFECTED PATIENTS].

The examination was carried out in the Moscow clinical infectious hospital No 2 concerning 102 patients with verified diagnosis "AIDS-infection" and seropositive according results of detection of anti-HIV-antibodies in blood serum. The study was organized to analyze rate ofcolonization of gums with virulent anaerobic bacteria in HIV-infected (polymerase chain reaction) and antibodies to HIV in gingival fluid (enzyme-linked immunosorbent assay). It is established that in HIV-infected patients, in scrape from gingival sulcus dominate anaerobic bacteria P. gigngivalis and A. ctinomycetemcomitans and in case of periodontitis--P. gingivalis and T. forsythia. The received data permits recommending the test-system "Multident-5" for polymerase chain reaction diagnostic. The reagents kit "Calypte®HIV-1/2"--for enzyme-linked immunosorbent assay gingival fluid. The results of polymerase chain reaction and enzyme-linked immunosorbent assay have no impact of concomitant stomatological (periodontitis, gingivitis) and somatic pathology.

[The use of self-adapting system files (SAF) for controlling microbial biofilms of root canals in the treatment of apical periodontitis]. / Primenenie sistemy samoadaptiruyushchikhsya failov (SAF) dlya bor'by s mikrobnoi bioplenkoi kornevykh kanalov pri lechenii apikal'nogo periodontita.

The aim of this study was to conduct a clinical and laboratory study and evaluate the effectiveness of endodontic root canal treatment using a self-adapting files system (SAF) in the complex treatment of patients with chronic apical periodontitis. 3% sodium hypochlorite solution was used as irrigation agent in all groups which included 20 patients treated with conventional manual tools, 21 patients receiving treatment with ultrasonic activation of irrigant and 26 patients treated with SAF system. Root canal biofilm structure was studied by scanning electron microscopy (SEM) using a Quantum 3D microscope (USA). Clinical efficiency of the root canal treatment was assessed by complications frequency a year after treatment. SEM revealed the presence of high levels of microbial contamination of dentine tubules in the apical portion of the tooth. In standard method group the percentage of re-treatment and surgery was higher than in the studied groups. Use of SAF irrigation system was associated with a decrease in the number of identified pathogens. However, the study revealed high resistance of Enterococcus spp., Porphyromonas gingivalis, Candida albicans to all types of endodontic treatment, so the improvement of methods of root canal microbial biofilms removing need to be continued.

[EXPERIMENTAL RATIONALE OF ENDODONTIC THERAPY OF CHRONIC FORMS OF PULPITIS- AND PERIODONTITIS USING PHOTO-ACTIVATED DISINFECTION AND ULTRASOUND TREATMENT].

AIM: Laboratory rationale of application of a complex effect of ultrasound treatment, and photo-activated disinfection in experiments in vitro using strains of anaerobic and microaerophilic microorganisms isolated from patients with chronic forms of pulpitis and periodontitis. MATERIALS AND METHODS: Microbiologic study of root canal was carried out using a bacteriologic method (2 species of obligatory-anaerobic and 2 -microaerophilic streptococci). Evaluation of the effect of ultrasound treatment in a complexwith a system of photo-activated disinfection on microflora of root canal was carried out using a bioreactor. Study of biofilms of the root canal system of a tooth in patients with chronic forms of pulpitis (52 individuals) and periodontitis (16 individuals) were carried out using scanning electron microscopy. RESULTS: The presence of an ample microbial bio- film in the ostiums of dentinal canaliculi and obturation of dentinal canaliculi in certain cases was established. Features of formation of growth curves for bacterial populations of oral microflora (Porphyromonasgingivalis, Fusobacterium nucleatum, Streptococcussanguinis, Streptococcus mutans) were described in the experimental part of the study as a result of a complex effect of photody- namic activation and ultrasound compared with control lacking those. CONCLUSION: The data obtained allow to conclude that complex effect significantly suppresses the growth or microbial populations of oral bacteria.

[STUDY OF COLONIZATION PROCESSES AND PERSISTENCE OF MICROORGANISMS IN ARTIFICIAL MATERIALS FOR MEDICAL USE].

AIM: Study processes of microbial colonization and persistence of microorganisms in polymer materials for medical use. MATERIALS AND METHODS: Samples (1 x 1 cm plates) of polymer plastics for production of removable dental prosthesis based on polyurethane and acryl were used, that were incubated with clinical isolates of Pseudomonas aeuruginosa, Staphylococcus aureus in Luria-Bertani broth nutrient media for 24, 48 hours and 7, 14 days and for 1, 5 and 3 months at a temperature of 37 degrees C. Dynamics of interaction process of microorganisms with polymer materials were studied using scanning electron microscope Quanta 200 3D (FEI Company, USA). The samples were fixated after incubation with 10% of neutral formaldehyde, dehydration with alcohols or acetone, typical for SEM, was not carried out, that allowed to conserve the native structure of the samples, including exo-cell matrix of biofilms. RESULTS: Electron-microscopical data on stages of interaction of bacteria with the surface of medical plastics were obtained. Biofilms were shown to be formed on abiotic surfaces and biodestructive changes of plastics appeared. A question on the possibility of prolonged persistence of pathogenic for human microorganisms in artificial prosthesis is discussed. CONCLUSION: The developed experimental model of formation of biofilm on abiotic surfaces could be the basis for carrying out studies directed on the fight with biofilms, by using SEM.

[PCR rationale for use of oral cephalosporins by oral surgery procedures].

The article presents the experience of PCR detection of DNA of pathogenic germs inducing odontogenic inflammation. Pus samples of 48 patients aged 18 to 68 years undergoing oral surgery because of apical periodontal lesions and pericoronitis. The results showed microorganisms associations revealed by PCR are sensitive to III generation cephalosporins. Effective oral regimen included 400 mg Ceftibuten once daily. The PCR results thus served as a rationale for use of oral cephalosporins by oral surgery procedures proved by clinical and immunological data in postoperative period.

[Features of adhesion of anaerobic periodontopathogenic bacteria and Candida albicans fungi to experimental samples of basis dental plastic depending on surface roughness and polishing method].

AIM: Study the main surface parameters of milled polyacrylic materials using atomic force microscopy and primary microbial adhesion of periodontopathogenic group bacteria and Candida albicans fungi taking into consideration the method of sample polishing. MATERIALS AND METHODS: Studied samples: mill-treated without polishing (control); ergobox polished; polished in dental laboratory conditions; polished by a rubber brush in dentists' office. Microbial strains belonging to periodontopathogenic species (clinical isolates) that had been isolated from periodontal pockets of periodontitis patients: Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus sanguis, C. albicans fungi were used for modelling experiments of primary adhesion of microbes to the material samples. RESULTS: S. sanguis had the highest degree of adhesion to polymer after milling, P. gingivalis, C. albicans--medium, F. nucleatum--low. A significant reduction of adhesion is observed during polishing in dental laboratory conditions or ergobox, less significant--during polishing in dental office. CONCLUSION: The data obtained allow to make a conclusion that the samples from polymer materials for preparation of prosthesis basis have varying degree of intensity of microbial adhesion of members of periodontopathogenic microflora and C. albicans fungi that depends on the polishing method, that accordingly determined the differences in colonization resistance against formation of microbial biofilm during polymer use in clinical conditions. . ,

[Gingival fluid cytokine profile in patients after dental implantation and by periimplantitis].

The study included 32 patients with complications which have developed 3 months to 14 years after dental implants placement. ELISA method was used to assess cytokines content in various sites of tooth alignment in patients with Herpesviridae and periodontopathogenics bacteria associated periimplantitis. The results prove the necessity for Herpesviridae and selected bacteria monitoring after dental implantation.

[Preventive antibacterial treatment in oral bone augmentation procedures].

The paper contains clinical and microbiological base for the use of Ceftibuten for prevention of inflammatory complications after bone augmentation and dental implant placement.

[Feasibility and assessment of combined anti-inflammatory, antibacterial and immunocorrective treatment in periodontal disease].

The article presents the experience of using medical dressings of combined action based on antibiotic Spiramycin and antiseptic Triclosan. Investigation and treatment of 79 patients with moderate chronic periodontitis proved the given drug formulation to be highly clinically effective for local treatment of periodontal disease.

[Biofilm formation on temporary dentures: correlation of primary adhesion, coaggregation and colonization].

Forty-six patients (20 male and 26 female, age 45-76) with temporary dental prosthesis were followed-up both clinically and microbiologically. The results of quantitative and qualitative assessment proved biofilm formed on acrylic resin to be potentially more aggressive because of high incidence and significant number of virulent germs as well as yeast-like Candida fungal. Laboratory polished polyurethane samples showed much lower colonization by virulent germs and Candida fungal. After biofilm stabilization PCR revealed twice-fold less incidence of virulent germs on polyurethane when compared to other tested materials.

[Expression of cytokines in dentogingival sulcus in patients after dental implantation and during development of peri-implantitis].

AIM: Determination of cytokine content in various areas of dentition in patients with peri-implantitis associated with parodontopathogenic bacteria species of I and II order. MATERIALS AND METHODS: 32 patients with complications that developed in 3 months to 14 years after installation of intraosteal dental implants were examined. Content of cytokines in various areas of dentition was determined by using solid phase enzyme immunoassay in patients with developed peri-implantitis associated with parodontopathogenic bacteria species of I and II order. Multiplex polymerase chain reaction was used for determination of parodontopathogenic bacteria marker DNA. RESULTS: Marker DNA of I order parodontopathogenic bacteria - Aggregatibacter (Actinobacillus) actinomycetemcomitans, Tannerella forsythia (Bacteroides forsythus), Porphyromonas gingivalis in peri-implantation tissues during implant rejection was detected with 34.4 - 75% frequency while II order (Prevotella intermedia, Treponema denticola, Parvimonas micros (Peptostreptococcus micros), Fusobacterium nucleatum/periodonticum etc.) - with significantly lower frequency. Total concentration of IL- 1beta, IL-4, IL-6, IL-8, TNFalpha, IL- 17A and INFgamma in contents of pathological pocket in the area of implants and the levels of each of them were significantly higher than in the contents of parodontal pockets, areas with stable implants and gingival fluid from areas with healthy teeth. Total interleukin content in the contents of pathological pockets in the area of rejected implants was significantly higher than in other studied areas. In the exudate of parodontal pockets of the preserved teeth it was 2.4 times lower, in the area of stable implants - 4.6 times lower, and in the areas with healthy teeth - 4.8 times lower than with rejected implants (p<0.05). CONCLUSION: The results of the study conducted allow to make a conclusion regard- ing reasonability of monitoring during dental implantation of parodontopathogenic microorganism strains and local cytokine response of the host organism with the objective of rational prophylaxis and therapy of inflammatory complications.