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.

[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.

[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.

[Sensitivity of microbial associations of periodontal lesions to antibacterial agents].

UNLABELLED: The aim of the study was the development of approaches to improve the effectiveness of antibiotic therapy in dental practice on the basis of determining the sensitivity of pathogenic microorganisms to antibiotics of different groups. MATERIAL AND METHODS: The study included determination of the sensitivity of the microbial complexes from wound exudate of periodontal pocket and apical abscess to macrolides, quinolones, penicillins, lincosamides and 5-nitroimidazole. A survey of dentists and dental clinics patients to identify the cause and frequency of use of antibiotics and to identify possible adverse reactions was also conducted. RESULTS: Dentists prefer macrolide antibiotics, protected penicillins, and fluoroquinolone combined with 5-nitroimidazole. All patients have taken antibiotics themselves at least once a year. Microbial complexes in patients with acute and exacerbated apical periodontitis in 79% of cases are susceptible to amoxicillin/clavulanic acid, to azithromycin - 52%, lincomycin - 36%, 5-nitroimidazole - 68%, ciprofloxacin - 73.7%. In patients with apical abscess high rates of resistance of microbial complexes to all types of antibiotics was revealed (33% for lincomycin 76,1% for ciprofloxacin, 28,6% for 5-nitroimidazole). Patients with moderate to severe periodontitis in 90.5% are sensitive to amoxicillin/clavulanic acid and azithromycin, in 62.4% to lincomycin. Sensitivity to ciprofloxacin was detected in 85.7% of patients, in 14.3% - moderate resistance.

[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.

[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.

[THE CHARACTERISTICS OF MORPHOLOGY OF BIOFILM OF PERIODONTIUM UNDER INFLAMMATORY DISEASES OF GUMS (CHRONIC CATARRHAL GINGIVITIS, CHRONIC PERIODONTITIS, CANDIDA-ASSOCIATED PERIODONTITIS) ACCORDING RESULTS OF ELECTRONIC MICROSCOPY].

The study was carried out to analyze morphology of biofilm of periodontium and to develop electronic microscopic criteria of differentiated diagnostic of inflammatory diseases of gums. The scanning electronic microscopy was applied to analyze samples of bioflm of periodont from 70 patients. Including ten patients with every nosologic form of groups with chronic catarrhal periodontitis. of light, mean and severe degree, chronic catarrhal gingivitis, Candida-associated paroperiodontitis and 20 healthy persons with intact periodontium. The analysis was implemented using dual-beam scanning electronic microscope Quanta 200 3D (FEI company, USA) and walk-through electronic micJEM 100B (JEOL, Japan). To detect marker DNA of periodont pathogenic bacteria in analyzed samples the kit of reagentsfor polymerase chain reaction "MultiDent-5" ("GenLab", Russia). The scanning electronic microscopy in combination with transmission electronic microscopy and polymerase chain reaction permits analyzing structure, composition and degree of development of biofilm of periodontium and to apply differentiated diagnostic of different nosologic forms of inflammatory diseases of periodontium, including light form of chronic periodontitis and gingivitis. The electronic microscopical indications of diseases ofperiodontium of inflammatory character are established: catarrhal gingivitis, (coccal morphological alternate), chronic periodontitis (bacillary morphological alternate), Candida-associated periodontitis (Candida morphological alternate of biofilm ofperiodontium).

[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.

[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.

[Study of virus content of Herpes viridae family in parotid secretion of patients with inflammatory-dystrophic diseases of salivary glands].

The study is devoted to disclose the interrelation between Herpes viridae family carriage and inflammatory-dystrophic diseases of salivary glands (SG). It was shown that inflammatory and dystrophic diseases of SG in observed patients run on the background of latent and active chronic herpes-virus infection. In the case the frequency of disclosure and content of antibodies to nuclear protein of Epstein-Barr virus in the peripheral blood serum in patients with different inflammatory and dystrophic SG diseases were statistically significantly higher and antibodies to simple 1st type virus and cytomegalovirus lower than in patients with different types of maxillofacial region pathology without SG involvement. The immediate contamination of parotid secretion by virus of Herpes viridae family was detected with the help of polymerase chain reaction in 18% of patients with SG diseases.

[Comprehensive microbiological and molecular-biological control of using laser technologies in ambulatory surgical practice].

Results of comparative use of different type lasers for surgical treatment of patients with medium severity level of periodontitis were presented: group 1--erbium laser "Waterlaser Millennium" (41 people); group 2--diode laser "LS-0,97 - IRE-Polyus" (25 people); group 3--carbon dioxide laser "СО2-laser LST - 20/01 Lantset" (21 people). Patients of the comparative group undergone a traditional surgical curettage (30 people). A PCR-diagnostic was made to all patients that were sent to ambulatory surgical treatment. It revealed carriage of 1 to 4 types of periodontium pathogenic bacteria that present ed threat from the point of view of infectious complications development in the postoperative period. After using laser technologies an increase of periodontium pathogenic species eradication frequency was registered (А. actinomycetemcomitans, T. forsithia, P. gingivalis, P. intermedia, T. denticola): up to 60, 58 and 60%--for erbium, diode and carbon dioxide correspondingly.