DOMINANT MICROBIAL ASSOCIATIONS OF ORAL CAVITY AT PERIODONTITIS AND FEATURES OF THEIR SENSITIVITY TO ANTIBACTERIAL DRUGS

in 100 % of cases within the oral cavity microbiota in patients with generalized periodon -titis. The most pronounced clinical symptoms of the disease and recurrent inflammation were observed with the persistence of the Staphylococcus genus bacteria association with the Enterobacteriaceae family bacteria and Pseudomonas bacteria genera , microscopic Candida genus fungi in the oral cavity. It was found that the fluoroquinolones and cefoperazone/sulbactam were the most effective against the isolates. Among the commercial preparations, Sangviritrin showed the most expressive antimicrobial activity and its antibacterial effect was registered on the clinical isolates of S. aureus . It is worth noting that the Sangviritrin showed the antimicrobial effect against an extra antibiotic-resistant isolates resistant to all phytopreparations and disinfectants used in the test. A high antimicrobial activity of Decasanum and Sangviritrin against the multi-antibiotic resistant strains, most by against Gram­positive bacteria, was established. The estab lished regularities determine the relevance of the antibiotic therapy taking into account the antibiotic susceptibility of the inflammatory process pathogens and the development of a comprehensive approach for correction of the opportunistic microbiota at generalized periodontitis. bacteria isolates were screened for a susceptibility to the following antibiotics: gatifloxacin norfloxa cin tetracycline azitromycin µ g clarithromycin µ g). The fungi isolates were screened for susceptibility to the following antibiotics: (in 6 mm diameter wells) with nystatin µ g ), itroconazole g) , fluconazole ketoconazole , voriconazole klotrimazole miconazole lower than that of Dioxydine and Dexasane. Chlorhexidine did not affect methicillin-resistant S. aureus and no antibacterial effect of chlorhexidine on the Streptococcus genus bacteria was detected.

Opportunistic microorganisms play a significant role in the development of inflammatory diseases, including generalized periodontitis. The growing tendency towards the formation of antibiotic resistant microorganisms causes the relevance of monitoring the microorganisms' sensitivity to antimicrobial drugs and the development of new approaches to the antimicrobial therapy. The purpose of this work was to determine the dominant microbial associations of the oral cavity in the conditions of generalized perio dontitis, to study their sensitivity to antibiotics, antiseptics and phytopreparations. The mic robial associations' study at generalized periodontitis was performed by bacteriological monitoring of the pathological material from the foci of the inflammatory process. Determination of the isolates' antibiotic susceptibility to antiseptics and phytopreparations was carried out by the disc diffusion method, in the agar. As test culture, the follo wing bacteria and yeast from the American Type Culture Collection were used: Candida albicans ATCC 885653; Staphylococcus aureus ATCC 25923; Escherichia coli ATCC 25922; Enterococcus faecalis ATCC 29212; Streptococcus pyogenes ATCC 19615; and Pseudomonas aeruginosa ATCC ATCC 27853. We also used clinical strains of bacteria and yeasts (S. aureus, K. rhinoscleromatis, H. alvei, E. coli, S. pyogenes, S. pneumoniae, C. albicans, C. glabrata isolated from the oral cavities of patients suffering from the inflammatory periodontium. We selected clinical strains with multiple resistance to antibio tics. The results of studies have shown that the opportunistic microorganisms dominated in 100 % of cases within the oral cavity microbiota in patients with generalized periodontitis. The most pronounced clinical symptoms of the disease and recurrent inflammation were observed with the persistence of the Staphylococcus genus bacteria association with the Enterobacteriaceae family bacteria and Pseudomonas bacteria genera, microscopic Candida genus fungi in the oral cavity. It was found that the fluoroquinolones and cefoperazone/sulbactam were the most effective against the isolates. Among the commercial preparations, Sangviritrin showed the most expressive antimicrobial activity and its antibacterial effect was registered on the clinical isolates of S. aureus. It is worth noting that the Sangviritrin showed the antimicrobial effect against an extra antibioticresistant isolates resistant to all phytopreparations and disinfectants used in the test. A high antimicrobial activity of Decasanum and Sangviritrin against the multi-antibiotic resistant strains, most by against Grampositive bacteria, was established. The established regularities determine the relevance of the antibiotic therapy taking into account the antibiotic susceptibility of the inflammatory process pathogens and the development of a comprehensive approach for correction of the opportunistic microbiota at generalized periodontitis.

INTRODUCTION
The inflammatory diseases of the periodontal tissues, including generalized parononitis, are multifactorial diseases, in whose etiology, the microbial factor occupies the key position [2,6,13]. At the first stages of the periodontitis formation, significant disorders of the periodontal tissues local immunity and anaerobic Gramnegative Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythensis bacteria play an important role, being involved in the periodontal pockets formation, the connective tissue destruction, and the alveolar bone resorption. The inflammatory infiltrate formation is accompanied by a variety of immuno pathologies.
Along with this, the cause of complications and recurrences of the inflammatory periodontal diseasse is the microbial cenosis restructuring and the opportunistic microorganisms domination within the microbial associations of the oral cavity. The colonization of the mucous membrane with transient and allochthonic microbiota representatives, frequently manifesting multiple resistance to antibiotics, leads to a permanent recurrence and chronic inflammatory process [4,15]. Due to this particular reason, some authors consider periodontitis that is an opportunistic infection, accompanied by the presence of opportunistic and pathogenic bacteria in the oral cavity [7,12,13]. It is also established that bacteria causing inflammatory periodontal processes can be a separate risk factor for cardiovascular, cerebrovascular diseases and premature delivery [8,11]. That is why, antibacterial drugs are widely used in treatment of the periodontal tissues [4,13,14].
At the same time, the everincreasing trend towards the formation of the antibiotic resistance, especially among the representatives of the opportunistic microbiota, including that in the biofilm [8,18], requires new approaches to both local and systemic treatment. Under such conditions, a permanent monitoring of the poly-antibiotic resistant microorganisms circulation, the study of their sensitivity to antibacterial drugs, the deve lopment of new integrated approaches and means for the oral microbiocenosis correction in the inflammatory process is relevant.
The purpose of the present study was to determine the dominant microbial associations of the oral cavity at generalized periodontitis, to clarify their sensitivity to antibio tics, antiseptics and phytopreparations.

MATERIALS AND METHODS
The isolates that caused periodontal inflammatory processes were isolated on the basis of the Dental Polyclinic at Uzhhorod National University; the antimicrobial activity was studied at the Microbiology Laboratory at the Department of Genetics, Plant Physiology and Microbiology, Uzhhorod National University.
We examined 180 patients diagnosticated for generalized periodontitis; the control group composed of 50 persons without such diagnosis. The biological material was collected from the mucous membrane of the inflammatory site using a sterile transport system (a test tube with gel and an applicator for biological fluids produced by FLmedical (Italy). The material was plated according to Gold on nutrient media: Sabourund Dextrose Agar, HiCrome™ Candida Differential Agar (Himedia) for the cultivation of microscopic fungi, hemolytic microflora, namely, the Streptococcus and Neisseria genera bacteria on the blood agar, Enterobacteriaceae genera bacteria -on Endo and Ploskirev agar (Farmaktiv, Ukraine), the Staphylococcus genus bacteria -on Mannitol Salt Agar (BiolifItalia), Enterococci were identified with Bile esculin agar (BiolifItalia ) We identified the bacteria and fungi based on macromorphology, micromorphology, physiological and biochemical tests using ENTEROtest, STREPTOtest, STAPHYLO test produced by Erba Lachema.
The antibiotic sensitivity of bacteria and microscopic fungi was identified by the disc diffusion method according to the accepted procedure (Order No. 167 of the MOH of Ukraine dated 05/04/2007; EUCAST (European Committee on Antimicrobial Susceptibility Testing).
The microorganisms' sensitivity to plantbased materials and disinfectants was determined by the standard agar diffusion test (with 8 mm diameter wells) [3]. The antibacterial properties was carried out according to the following criteria: absence of a growth retardation zone of 10 mm, indicating that the microorganisms are not sensitive to the sample inserted into the well; that of 10-15 mm indicates a weak sensitivity; 15-25 mm means sensitivity; more than 25 mm -high sensitivity.
The data obtained were expressed as mean ± standard deviation (SD) of three measurements. The Tukey's test was applied for comparisons of mean values, differen ces were considered reliable at p<0.05. Statistical analysis and comparisons among the means were carried out using Microsoft Excel 2013. The parameters calculated alongsi de with the basic variation were: average and standard deviation; minimum and maximum coefficients of variation; and the inhibition zones size frequency.

RESULTS AND DISCUSSION
The total of 389 opportunistic microorganisms strains were isolated from 180 patients with persistent inflammatory process at generalized periodontitis. The Staphylococcus genus bacteria were isolated from the inflammatory site in 73 % (131/180) cases and were represented by four species: S. aureus, S. haemolyticus, S. saprophyticus, S. epidermidis. In 53 patients S. aureus was isolated, 20 of them being methicillinresistant. In 44 patients, S. haemolyticus was isolated.
The total of 45 microscopic fungi strains of the Candida genus were isolated, 38 of them belonging to С. albicans, 5 strains -to C. glabrata, 2 -C. cruzei. In patients with generalized periodontitis, the following representatives of indigenous microbiota were isolated: nonpathogenic streptococci S. sanguis, S. mitis, S. salivarius. In 57 % cases the Lactobacillus genus bacteria were isolated. By studying the microbiota of mouth cavity in control group patients, we ascertained presence of the nonpathogenic Streptococcus spp.; lactobacilli were identified in 91.5 % of the reviewed material and only in 57 % of patients suffering from the periodontitis. The microbiota of the healthy gum tissues was characterized by a low population level of microbial populations of opportunistic microorganisms (S. saprophyticus, S. epidermidis) -22 %. However, at chronic generalized periodontitis, the opportunistic asso ciations prevailed.
A high degree of antibiotic resistance in the microorganisms isolated from the inflammatory process foci with generalized periodontitis was shown -48 % of isolates were resistant to at least 7 antibiotics. Thus, 3 isolates of K. rhinoskleromatis were resistant to 30 antibiotics. All the isolates were resistant to ampicillin, erythromycin, tetracycline.
The study results on the isolates' antibiotic susceptibility showed that 57.65 % of isolates were susceptible to amoxicillin/clavulanate, 5.3 % were moderately sensitive and 37.05 % were resistant.
Isolates in the amount of 85.29 % showed resistance to cephalosporins of the 1st generation. Sensitivity to cephalosporins of the second generation has been established: to ceftriaxone -61.76 % and to cefuroxime -55.29 % of cultures. It was shown that 97 % of isolates were susceptible to cefoperazone/sulbactam.
It was established that 73 % of all isolates were susceptible to fluoroquinolones, in particular to fluoroquinolones of the second generation: to ofloxacin -41.76 % of isolates; to norfloxacin -68.23 %; to lomefloxacin -32.35 %; to ciprofloxacin -68.82 %; to fluoroquinolones of the third generation: to levofloxacin -75.29 %, and to the forth gene ration fluoroquinolones (gatifloxacin) -87 %.
The opportunistic microorganisms were susceptible to the cabopenems: 80 %to meropenem and 58.23 % -to imipenem.
Of the 170 isolates, 15.29 % were sensitive to azithromycin, 30 % were moderately susceptible and 55.29 % were resistant. 30 % of cultures were sensitive to the semisynthetic clarithromycin macrolides.
The microscopic Candida genus fungi was resistant to fluconazole, 6 strains showed sensitivity to itraconazole, 10 -to clotrimazole.
The conducted studies have shown that Dexasan antiseptic exhibited a wide spectrum of antimicrobial activity (see Figure). In particular, the sensitivity of all bacteria involved in the experiment as clinical isolates and typical cultures has been established. The highest levels of antimicrobial activity were recorded for the Staphylococcus genus bacteria, including methicillinresistant strains. However, the antimycotic action of Dezasan on Candida species in the drug dose selected by us was not detected.
High antibacterial effect was observed as a result of the Dioxydine action, but the parameters of growth retardation zones varied greatly from 30.33 ± 0.58 mm to the clinical strain of S. aureus to 17.33 ± 0.33 c to S. aureus MRSA. High antibacterial acti vity of dioxydine on S. pneumonia has also been established. The bactericidal activity of the drug against E. faecalis and K. rhinoscleromatis was not detected. Moderate activity of the dioxydine against E. coli has been established.
The moderate sensitivity of the Staphylococcus genus bacteria to chlorhexidine is shown, but it is much lower than that of Dioxydine and Dexasane. Chlorhexidine did not affect methicillin-resistant S. aureus, and no antibacterial effect of chlorhexidine on the Streptococcus genus bacteria was detected. The studies have shown a high level of the antimicrobial activity of the Sangviritrin drug, possessing a high antibacterial effect on Grampositive microorganisms, a moderate effect on Gramnegative bacteria and a weak antimycotic activity ( Table 1, Table 2). The Chlorophyllipt phytopreparation demonstrated high in vitro efficacy towards the Staphylococcus genus bacteria, more towards typical strains than to clinical ones, and to E. faecalis, weak and moderate activity towards E. coli.
The Eucalyptus tincture showed a high and moderate activity towards the isolates. No antimicrobial activity of the Mint rinse was detected, and low and moderate activity of the "Salvia" tincture towards the Staphylococcus and Streptococcus genera bacteria was observed. The Rotocanum drug produced a moderate and low antimicrobial effect on isolates involved in the experiment, except for E. coli and S. aureus (MRSA).
At chronic persistent inflammatory process, the opportunistic microorganisms domi nate in the oral cavity of patients with generalized perio dontitis.
The obtained results are consistent with the data of other researchers [8] who established the domination of the Staphylococcus genus bacteria in the microbiota of the oral cavity in patients with generalized periodontitis. The authors also identify the S. aureus, S. epidermidis, Bacteroides spp., Actinomyces spp., Candida spp., Pseudomonas aeruginosa and Streptococcus spp. microorganisms, as the most important agents  Table 1.  Примітка: дані у стовпці, позначені різними літерами, достовірно відрізняються за P<0,05 згідно з Тукі тестом; "" -затримки росту немає causing the development of generalized periodontitis and the role of the above microorganisms proteolytic enzymes, including collagenolytic enzymes, hyaluronidase, chondroitin sulfatase in the disintegration of collagen, the main protein of the periodontal tissue. The authors also report high sensitivity of the opportunistic microbiota to clinda mycin and ofloxacin.
Colonization of the oral cavity with pathogenic bacteria can be an antecedent and, in its turn, can lead to other diseases [1]. Diseases of the periodontal tissues contribute to the oropharyngeal colonization by potential respiratory pathogens, including Enterobacteriaceae (K. pneumoniae, E. coli, Enterobacter sp., P. aeruginosa, and S. aureus).
The persistence of opportunistic microorganisms in the oral cavity creates preconditions for the development of systemic diseases, especially, this correlation is established in the elderly persons [1,2].
The domination of opportunistic microorganisms in the oral cavity in conditions of generalized periodontitis causes the need for antibacterial therapy. The literature data report that according to the dental practitioners' survey, amoxicillin (73.58 %) is the most frequently prescribed as an antibiotic systemic treatment, 36.47 % of dentists recommend lincomycin to patients, and fluoroquinolone preparations (ciprofloxacin) are prescribed by 30.18 % of dental practitioners, doxycycline -by 17.61 %, clarithromycinby 5.03 % [1]. Our studies showed high sensitivity of the opportunistic microorganisms, isolated from the inflammatory foci, to fluoroquinolones, however, their high resistance to penicillins, macrolides, lincomycin, doxycycline was established.
It is shown that most microorganisms causing complications of dental implantation are resistant to penicillins, macrolides, lincosamines [2]. A significant increase in the level of antibiotic resistant strains of microorganisms in conditions of periodontal inflammatory diseases, may be due to the presence of bacteria in the composition of biofilm. It has been proved that the resistance of bacteria to antibiotics within the biofilm is seve ral orders greater [4,6,11,17].
At the same time, the auxiliary or alternative means of the opportunistic microbiota correction is the use of local antiseptics and phytopreparations to potentiate the action of antibiotics, and in benign cases, an alternative to their application [5,8]. We have established low efficacy of the antimycotic drugs, particularly their local effects. Meanwhile, in our previous studies, high activity of essential oils [9,10,16] and phytopreparations to isolates of opportunistic infections pathogens, including microscopic fungi of the Candida genus was established.

CONCLUSION
At generalized periodontitis, the domination of conditionally pathogenic microorganism associations in the microbiote of the oral cavity was observed. Association of the bacteria of Staphylococcus genus and Enterobacteriaceae family accompanied the most complex recurrent inflammatory processes. High percentage of the antibiotic resistant isolates is shown. It was found that the most effective against the isolates were fluoroquinolones and cefoperazone/sulbactam. Among antiseptics, Decasanum demonstrated a wide range of the antibacterial activity. The high antimicrobial effect was demonstrated by Sangviritrin.
The application of a comprehensive, differential approach to the correction of microbiota in the oral cavity at generalized periodontitis is grounded. Taking into account the dominant associations and their sensitivity to antibacterial drugs, including phytopreparations, which, in addition to antimicrobial activity, do not violate the composition of the indigenous microbiota.

ACKNOWLEDGMENTS
The present study is a fragment of the research project at the Department of Gene tics, Plant Physiology and Microbiology of Uzhhorod National University "Research of genetic, physiological and biochemical mechanisms of various organization level biological systems adaptation in the anthropogenic loading conditions", No. 0115U003902.

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.