RU2661609C1 - Tongue microbioma as a prognostic model for determining the seeding of cariesogenic bacteria of streptococcus mutans of dental hard tissues in young children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine – dentistry/pediatric dentistry and microbiology/bacteriology. Method for determining the dissemination of cariesogenic bacteria by Streptococcus mutans of hard tooth tissues in young children involves the collection of biomaterial from the entire surface of the mucous membrane of the middle third of the tongue with sterile stupor, further the biomaterial is applied to standard test strips “Dentocult SM Strip Mutans” with a square end and a rough surface, they are placed in a selective nutrient medium for cariogenic streptococci, after incubation, the degree of colonization by streptococci of the microbiota of the mucous membrane of the tongue back is estimated, including their number in classes from 0 to 3, respectively from CFU < 10⁴/ml to CFU > 10⁶/ml, after referring to the class of dissemination of S. mutans microbiomas of the tongue, determine dissemination by them of the biotope of the lingual surface of the baby teeth, considering that the quantity/density/seeding/titer of S. mutans in the microbiome of the tongue statistically reliably corresponds to the Streptococcus mutans titer in the dental plaque of the tongue surface of the baby teeth.

EFFECT: invention can be used to determine the titer of cariesogenic bacteria Streptococcus mutans in dental plaque on the model of the tongue microbioma in young children.

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Description

The invention relates to the field of medicine - dentistry / pediatric dentistry and microbiology / bacteriology and can be used to determine the titer of cariogenic bacteria Streptococcus mutans in plaque on a model of the tongue microbiome in young children.

Acceleration of the physical development of children leads to early eruption of both temporary and permanent teeth. The tooth enamel “does not have time” to be saturated with macro- and microelements, and after eruption, teeth that are at the stage of structurally functional immaturity are highly susceptible to aggressive effects of cariogenic factors (Kiselnikova L.P., Zueva T.E., Kruzhalova O.A. Caries of temporary teeth in young children: substantiation of etiopathogenetic approaches to preventive treatment // Dentistry for children and prevention. 2000. No. 2. P. 19-22; Korchagina V.V. Treatment of dental caries in young children. M .: MEDpress- inform. 2008. 95 p.). Due to the fact that the susceptibility of enamel to the effects of acids during the period of incomplete mineralization is greatest, in the first 2-3 years after eruption there is a high prevalence of focal demineralization, the likelihood of which increases with insufficient hygienic care of the oral cavity and the consumption of a large amount of carbohydrates (Korchagina VV Treatment of dental caries in young children. M: MEDpress-inform. 2008. 95 p .; Kuzmina EM. Prevention of major dental diseases. M: Medicine, 1997. 45 p .; Kaste L.M., Drury T.F., Horowitz A.M., Beltran E. An evaluation of NHANES III estimates of early childhood caries. J Public Health Dent 1999; 59: 198-200; Berkowitz RJ. Cause, treatment and prevention of early childhood caries. J Can Dent Assoc. 2003; 69: 304-7).

The period of physiological maturation, that is, the enamel mineralization takes more than 2 years, and throughout the entire period of mineral maturation, especially during the first year after teething, the child’s teeth need careful and effective care (T. Kharitonova, S. N. Lebedeva ., Kazakova LN Early prevention of dental caries in children // Saratov Scientific and Medical Journal. 2011. V. 7, No. 1 appendix. P. 260-262).

In early childhood caries, temporary teeth are affected almost immediately after eruption. The first carious lesions are usually found on the vestibular surface of the maxillary incisors in the cervical region in the form of areas of chalk-like color. The carious process is characterized by the speed of the course, spreading in width, multiple lesions of the teeth in the order of their eruption, except for the incisors of the lower jaw. A rare lesion of the lower incisors with this pathology is explained by the best self-cleaning capabilities due to the position of the tongue and abundant washing with saliva (Kuzmina EM. Prevention of dental diseases. M .: Medicine. 2001. 32 p.).

The accumulation of soft bacterial plaque on the surface of the teeth interferes with the physiological maturation of the enamel of temporary teeth, as it prevents the entry of macro- and microelements from saliva. Plaque microorganisms are a key etiological factor in the development of the carious process. Bacteria in the biofilm are always metabolically active, causing fluctuations in the pH of saliva, they produce organic acids, acting on immature enamel progressing tooth decay in young children (Seow KW. Biological mechanisms of early childhood caries. Community Dent Oral Epidemiol. 1998; 26 (1 Suppl): 8-27).

The earlier risk factors are identified, eliminated, or weakened, the smaller the increase in caries is observed in children. Prevention of dental caries in children is the key to dental health for life. Preservation of healthy temporary teeth until the physiological shift will avoid many problems with permanent teeth in the future.

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derung. 2009; 4. - P. 119-123).Many authors noted that measures aimed at reducing the level of caries in children, consisting in early prevention and treatment, the prevalence and severity of the carious process in various regions of our country, are still at a high level (Kiselnikova L.P., Tokareva A.V. Early childhood caries and its complications 9th Congress of the European Academy of Pediatric Dentistry, Croatia, Dubrovnik, may 29 - june 1, 2008, p. 107; Skripkina G.I., Smirnov S.I. A model for the development of the carious process in children // Pediatric Dentistry and Prevention.-2012. - No. 3. - S. 3-7.). At the same time, the decay rate of specifically deciduous teeth among children of early and preschool age increased almost 2.5-3 times (Kharitonova T.L., Lebedeva S.N., Kazakova L.N. Early prevention of dental caries in children // Saratov Scientific -medical journal. - 2011. Volume 7 - No. 1. Appendix. P. 260 .; Shakovets N.V. et al. Risk factors for the occurrence of early childhood caries according to the results of three countries // Pediatric Dentistry and Dental Disease Prevention: Sat. Proceedings of the VII Scientific and Practical Conference with International Participation / Mos . Islands, May 16, 2011 under the editorship of Professor Kiselnikova LP, Drobotko LN - S. 213-215).. According to foreign authors, the greatest increase in the prevalence and intensity of caries is observed in the age category from 2 to 5 years (Borutta A., Wagner M., Kneist S. Early Childhood Caries: A Multi-Factorial Disease // OHDMBSC; Splieth C.H. , Treuner A., Berndt C. Oral health in toddlers.

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derung. 2009; 4. - P. 119-123).

Caries is an infectious disease that does not threaten the life of a child, but significantly affects the functional properties and growth of the body, and reduces the quality of life (American Academy of Pediatric Dentistry. Symposium on the prevention of oral disease in children and adolescents. Chicago, I11, November 11-12 2005: Conference papers. Pediatr Dent 2006; 28 (2); 196-198).

In Belterami G (ed). La

infantile. Marseille: Leconte 1952).Due to the high prevalence of this pathology, a special term has been proposed in foreign literature: “Early Childhood Caries”, which describes the presence of one or more carious, removed due to complicated caries or a sealed surface in any temporary tooth in children aged 71 months and earlier (Milnes AR Description and epidemiology of nursing caries. J Public Health Dent 1996; 56: 38-50; Tinanoff N, Kaste LM, Corbin SB. Early childhood caries: positive beginning. Comm Dent Oral Epidemiol 1998; 117-119). Early caries has long been recognized as a clinical syndrome, described by Belterami in the 1930s as “Les dents noire de tout-petits” (Belterami G. Les dents noires de tout-petits.

In Belterami G (ed). La

infantile. Marseille: Leconte 1952).

For such a multifactorial disease as early childhood caries, conducting prophylactic preventive measures does not make it possible to hope for a high end result, it is necessary to look for other, modern approaches to the prevention of dental caries and requires a mandatory determination of an individual's individual predisposition to this disease (Puzikova O.Yu., Omsk , 1999). Along with mass prophylactic measures, individualized prophylaxis is carried out, based on prenosological diagnosis and prognosis of diseases of the maxillofacial region (Laurisch L., ZWR., 1990; Melnik A.I., abstract of dissertation, 1991; Li Y., W. Wang J Dent. Res., 2002; M. Fontana et al., J. Dent. Res., 2011). Analyzing the above, the issue of diagnosing the risk of developing early childhood caries in the process of a comprehensive analysis of the most significant clinical and subclinical parameters and developing individual approaches to treatment and prevention methods of correction is relevant.

To date, the leading role of microflora in the occurrence of dental caries has been proven (Kneist S., Maslak E., Tsare R., Berzina S., Skrivele S., Terekhova T., Shakovets N., Wagner M., de Mura - Sieber V. , de Mura R., Berutta A. Biological and social risk factors for early childhood caries // Modern Dentistry. -2011. - No. 1. - P. 62-65).

Plaque, due to the accumulation of microorganisms, is able to convert carbohydrates from food into acid and lead to the formation of a focus of enamel demineralization and the subsequent formation of a carious cavity. After 6 hours after a thorough brushing, you can determine the presence of plaque, which quickly forms when you eat soft foods and sticky carbohydrates. Sucrose accelerates plaque formation and is the most significant aggressive factor, as it converts non-cariogenic / anticariogenic products to cariogenic, promotes an increase in the proportion of Streptococcus mutans and lactobacilli, while reducing Streptococcus sanguini (Douglass JM Response to Tinan off and Palmer: Dietary determinants of dietary recommendations for preschool children. J Public Health Dent 2000; 60 (3): 207-209; Paes Leme, H. Koo, C. M. Bellato, G. Bedi, and JA Cury. The Role of Sucrose in Cariogenic Dental Biofilm Formation- New Insight. J Dent Res 2006, 85 (10): 878-887).

The rapid growth of the plaque is observed during sleep, its maximum formation is carried out after 30 days. Plaque plays a major role in the activation of the life of microbes, creating conditions for acid formation, enzymatic activity and other metabolic processes and products (E.V. Borovsky, V.K. Leontyev, Oral Biology. M. Medicine, 1991. 304 p.; Grokholsky A.P. Kodola N.A., Tsentilo T. Dental deposits: their effect on teeth, peridental tissues and the body, Kiev: Zdorovye, 2000. 160 pp .; Levitsky A.P., I.K. Mizina. , 2nd ed., Kiev.Health, 1987. 80 pp .; Leontyev V.K., V.T. Shestakov, Assessment of the main directions of development of dentistry, M .: . Power Book, 2007. 279 p .; Solov'eva AM pH of dental plaque and saliva role in its new normalization in dentistry 2000. -... №3 (94) pp 33-39).

The microflora that inhabits the human body in the biotope can either be in a free state or in a bound state, forming a biofilm. Unfixed / planktonic microflora peels off the surface of the biofilm and is released into the environment with human secretions, for example, with saliva, with feces. The microflora that forms the biofilm closes the receptors from external encroachment, while freely living microorganisms through their metabolic products enter into competitive interactions with foreign microflora (Costerton JW, Stewart PS, Greenberg E.P. Bacterial biofilms: a common cause of persistent infections // Science. -1999. - No. 284. - C. 1318-1322).

Biofilm is a special form of microflora organization in the human body. It is a well-interacting community of microorganisms, consisting of bacteria of one or several species, occupying sensitive receptors in the macroorganism and colonizing them, and also separated from the external environment by a structure that is a derivative of the vital products of microorganisms and tissue cells on which they adhere. According to the conceptual model of dental biofilm according to Jill S. Nield-Gehrig (2003), it consists of bacteria; microcolonies, aggregates of microcolonies of bacteria, extracellular polysaccharide matrix of EPS substance, epithelial cells, components of saliva and food, blood cells. Biofilm associated with dental health and periodontal / Health-associated dental plaque. A microbiological portrait of a healthy biofilm is presented: S. sanguinis, S. mitis, S. orates, S. salivarium, Veillonella, Actinomycetaceae, Haemophilus, Bacteroides. The biofilm associated with caries, periodontitis, peri-implantitis / Disease-associated dental plaque is represented by a violation of microbial homeostasis: the dominance of acidogenic and acid tolerant Streptococci spp. and Lactobacillus spp., the loss of the dominant main symbiont position and an increase in the representation of transient microflora (O.S. Gileva, G. Masis, conference proceedings. “Ahead of the future”, M. Endodontia today, 2012).

Special studies have shown that in a biofilm differently, in comparison with pure cultures of bacteria, their numerous physiological and biological processes occur (O'Toole GA, Kaplan N.V., Kolter R. Biofilm formation as microbial development // Annu. Rev Microbiol. - 2000. - No. 54. P. 49-79). The community organizes a unified genetic system establishing behavioral forms for biofilm members, determining their food / trophic, energetic, and other relationships between themselves and the outside world (Slots J, Jorgensen M.G .: Effective, safe, practical and affordable periodontal therapy: where are we going, and are we there yet? Periodontology 2002). The latter received a special name: “social behavior of microorganisms” - “quorum sensuum” (Babin VN, Domaradsky IV Biochemical and molecular aspects of the symbiosis of man and his microflora // electronic version of Ross, chemical journal. - 2006. - No. 6 .., Gunzburg A.L., Ilyina TS, Romanova Yu.M. “Quorum sensing” or the social behavior of bacteria // ZhMEI - 2003. - No. 5. - P. 86-93).

Pathogenic bacteria do not show aggressiveness against the host organism until the degree of their virulence reaches the necessary level in order to effectively overcome the defense of the host organism. In vitro antibiotic resistance of the biofilm is 500-1000 times higher than that of individual bacterial colonies (Allais G. Oral Biofilm, New in Dentistry. 2006).

Among the microflora that inhabit different ecological niches of the human body, indigenous / autochthonous microflora and alien, that is, introduced into the body from the outside are distinguished (Bondarenko V.M. Human microflora: norm and pathology // electronic version of the scientific and information journal "Science in Russia". - 2007. - No. 1, Osipov G.M.

Invisible organ - human microflora. M., 2008). The composition of indigenous microflora is determined by the characteristic types of microorganisms, the number of which is small, but the concentration is always significantly expressed. The detection rate of S. mutans, S. salivarius, S. mitis in saliva and gingival pockets is 100.0% of cases, and the concentration of each of them reaches 10 ⁵ -10 ⁸ in 1 ml. saliva (Borovsky E.V., Leontiev V.K. Biology of the oral cavity. - M.: Medical book, 2001. - 304 p.).

It is the characteristic groups of microorganisms that determine the mechanisms of stability formation in the biotope. For them, the definition of "basic symbiotic unit", i.e. that is the smallest number of species that can coexist in competitive conditions and in which none of them could survive on their own in these environmental conditions (B. Shenderov Medical microbial ecology and functional nutrition: 3 t. T. 1. The microflora of humans and animals and its functions. - M.: GRANT, 1998. - 288 p.).

The most complex in composition are the microbiocenoses of the oral cavity, nasopharynx and colon (Yankovsky D.S. Composition and functions of microbiocenoses of various human biotopes // electronic version of the All-Ukrainian scientific and practical journal “Women's Health”. - Kiev, 2003. - T. 16 , No. 4). Microbiocenoses of the skin and nasal passages are simpler (B. Shenderov Medical microbial ecology and functional nutrition: 3 vol. T. 1. The microflora of humans and animals and its functions. - M.: GRANT, 1998 - 288 p., Yankovsky D. S. Composition and functions of microbiocenoses of various human biotopes // electronic version of the All-Ukrainian scientific and practical journal “Women's Health.” - Kiev, 2003. - T. 16, No. 4).

Colonies of bacteria have the property of adhesion to organic structures and form a plaque, which was recently called a bacterial plaque. (Asikainen S, Chen C: Oral ecology and person to person transmission of Aa and Pg. Periodontology 2000, Rateischak K.H., Wolf H.F .: Parodontologie. 2. Aufl. Thieme 1989). In the modern interpretation, bacterial plaque is also called “biofilm, which is a specialized bacterial ecosystem that ensures the viability and preservation of its constituent species of microorganisms and increases the general population” (Caufield PW, Cutter GR, Dasanayake A. P.: Initial a cquisition of mutans streptococci by infants : evidence for a discrete window of infectivity. J DentRes 1993, Leus P.A. Communal dentistry, 2000). Bacteria can adhere to various surfaces of the oral cavity, including saliva-coated teeth, epithelium of the cheeks, gums and tongue, and subsequently colonize them. If these surfaces are already colonized, the ability of bacteria of different species to adhere to each other in the process of cohesion is of particular importance. Such an interaction between bacteria leads to the formation of biofilms heterogeneous in composition and the formation of food relationships between microbes.

Most of the oral streptococci that inhabit various biotopes of the upper respiratory tract and oral cavity belong to vermin streptococci. They do not contain group-specific carbohydrates / C-polysaccharides in the cell wall and therefore are not included in the serological classification (R. Lansfield, 1933). Various types of oral streptococci selectively colonize certain areas: S.mitior - buccal mucosal epithelial cells; S. mutans - tooth enamel. The number of oral streptococci susceptible to significant changes, as in the intestinal microbiocenosis, is an informative indicator and reflects the degree of homeostatic disorders (Razzhivin, A.P. Dysbiotic conditions in children: clinic, diagnosis and treatment study guide. N. Novgorod: NGMA. - 1999 . - 123 p.).

Microflora is the most important structural component of the multicomponent oral system, an indicator of dental and systemic health, and the state of the environment (OS Gileva, conference materials. “Ahead of the future,” G. Masis, M. Endodontia today, 2012). Normal oral flora takes part in the formation of plaque or plaque. Based on the data of numerous studies, the composition of microflora, the plaque is characterized by pronounced heterogeneity.

It is known that more than 800 species of the studied microorganisms inhabit the oral cavity of a healthy person, preserving the ecosystem and not disrupting homeostasis (Slavkin, 1999, Tchaikovskaya I.V., 2002, Zelenova E.G. et al., 2004, Tets V.V., 2008). Gram-positive streptococci of the Streptococcus mutans species, which initiate the formation of plaque as a biological film on the tooth surface, which, due to coadhesion and accumulation of other representatives of the pathogenic flora (Kuznetsov E.A., Tsarev V.N., etc.), play a special role in the occurrence and development of caries. , 1996; Burt and Pai 2001, Berkowitz, Tchaikovsky I.V., 2002, RJ Causes, 2003, Tets V.V., 2008, Skripkina G.I., 2014). The hypothesis about the role of streptococci in the development of caries has been accepted in the USA for practical guidance since 1960 after studies conducted in the country since the 40s of the XX century (Shafer W.G., A textbook of oral pathology, 1974)

Streptococci account for a significant proportion of dental plaque microflora, up to 80% of the total number of oral microorganisms (Maksimovskiy Yu.M. et al., 2009), which is consistent with the data of foreign authors, reporting the presence of acid-forming streptococci in 86% (Weiilherell IA Composition of dental enamel // Brit. Med. Bull. 1975. - Vol. 31, No. 2. - P. 1415-1420). The most important factor in the virulence of Streptococcus mutans is their prosperity in an acidic environment (Ovrutsky G.D., Leontiev V.K., 2010). By metabolizing carbohydrates, S. mutans intensively produce lactic acid, many times faster than other microorganisms of soft plaque. Each sugar intake causes a “metabolic explosion” in the oral cavity (Leontyev VK, Pakhomov GN Prevention of dental diseases. M. 2006 - 416 p.). These properties allow streptococci to be the first to populate various biotopes of the child's oral cavity (V.S. Kramar et al., 1992). The main biotopes of the oral cavity are the mucous membranes, the back of the tongue, gingival sulcus, oral fluid and plaque (Zelenova E.G. Zaslavskaya M.I., Salina E.V., Rassanov SP. Microflora of the oral cavity: norm and pathology: Textbook . - N. Novgorod: Publishing house of the Novosibirsk State Medical Academy, 2004 .-- 158 p.). S. mutans is selectively adsorbed on the hard surface of the teeth. There are conflicting data on the streptococcal contamination of individual biotopes (Zelenova E.G. et al., 2004, Skripkina G.I., 2014).

It was previously believed that Streptococcus mutans were infected between 19 and 31 months old through the so-called discrete infection window (Caufield PW, Cutter GR, Dasanayake A.P .: Initial a cquisition of mutans streptococci by infants: evidence for a discrete window of infectivity J DentRes 1993). But it has now been proven that infection with cariogenic species of S. mutans and their colonization in grooves of the tongue are possible even before teething (Berkowitz 2003), which confirms the transmission of Streptococcus mutans from mother to child (Kuzmina EM Dental disease prevention: training allowance. Moscow, 2001; EM Kuzmina. Prevention of dental caries as the most important aspect of preserving the dental health of children. // Russian pediatrician. Zhurn. - 2006. - No. 6). At the same time, Tanzer writes that hard surfaces are needed to colonize Streptococcus mutans, as evidenced by the rapid appearance of S. mutans in infants before teething when obstructors are used to correct cleft lip (Tanzer, Livingston et al, 2001). Studies (Thorhild I, Lindau-Johnson B, Twetman S, 2002, Berkowitz, 2003, Shakovets H.V., 2009, Caufield, Ratanapridakul et al, 1988, Loveren, Buijsetal, 2000; Kuzmina E.M., I . I. Lysenkova, Prevention of dental caries as the most important aspect of preserving the dental health of children, // Russian Pediatrician, Journal of 2006 - No. 6; Kiselnikova LP Individual prevention of caries in school-aged children / Clinical Dentistry. - 2006 ).) also confirmed the horizontal transmission of cariogenic streptococci: children attending the same kindergarten have identical strains of bacteria in their saliva (Berkowitz, 2003), and children who are at home have identical strains of streptococci and their titer with their mother and father (Seow K W., Biological mechanisms of early childhood caries. Community Dent Oral Epidemiol. 1998; 26 (1 Suppl): 8-27; Caufield PW , Griffen A L. Dental Caries. An infectious and transmissible disease. Pediatr Clin North Am. 2000; 47: 1001-19). Streptococcus mutans is one of the leaders of the bacterial community in the etiology of caries (Khavkin A.I., Ippolitov Yu.A., Aleshina E.O., Komarova O.N., Microbiota and oral diseases, 2015, 78-81).

Thus, the earlier the child’s oral cavity is colonized by S. mutans, the higher the chance that immature enamel of recently erupted teeth will be coated with plaque, the properties of which will depend very much on the food consumed (Alaluusua S, Malmivirta R. Early plaque accumulation, a sign for caries risk in young children. Community Dent Oral Epidemiol 1994; 22: 273-276). The food content of easily fermentable carbohydrates will be high, then S. mutans will be in symbiotic interaction with Lactobacillus. spp., synthesizing extracellular polysaccharides conducive to increased stability of the tooth plaque matrix (Tinanoff N, Kanellis M J, Vargas C M. Current understanding of the epidemiology mechanisms, and prevention of dental caries in preschool children. Pediatr Dent 2002; 24: 543-55 ) At the same time, there is a clear link between the high incidence of Streptococcus mutans in mothers and the risk of massive contamination of their children, and the higher the incidence of caries by 4 years of life (American Association of Pediatric Dentistry (AAPD). Dental care for your baby. 2009).

Medical monitoring of non-specific biofilms is a simple and common effective tool in the treatment of caries and periodontal diseases (Kuzmina EM. Dental disease prevention: a training manual. - Moscow, 2001., Socranssky SS, Haffajee AD: Dental Biofilms: difficult the rapeutictargets. Periodontology 2002).

Treatment should cover the entire surface of the oral cavity and the adjacent surface of the pharynx, and not just the area of teeth and periodontium (Theilade E. Factors controlling the microflora of the healthy mouth. In: Human microbial ecology. Hill MJ, Marsh PD Boca Raton, FL, CRC Press 1990). The main goal of regular mechanical plaque removal is to reduce the total number of bacteria and modify the composition of the biofilm.

The spread of plaque on the tooth surface occurs from the interdental spaces and gingival grooves; growth of colonies is similar to the development of the latter on a nutrient medium (Asikainen S, Chen C: Oral ecology and person to person transmission of Aa and Pg. Periodontology 2000, Kuzmina E. M. Prevention of dental caries as an important aspect of preserving children's dental health / I. I. Lysenkova // Russian Pediatric Journal, 2006, Netuschil L: Biofilm alsorganisation form der plaque. Prophilaxe Dialog 2004; Wilton JMA, Bampton JLM, Hurst TJ: Interleukin-IB and Ig G subclass concentration in gingival crevicular fluid from patinets with a history of destructive periodontal disease. Journal of Clinical Periodontology, 2005).

Predictive methods, which are aimed at the initial prevention of the carious process in children, consider mainly subjective criteria in collecting an anamnesis: aspects of feeding, physiological status and physical activity, carbohydrate intake, socio-economic standard of living, pregnancy, child morbidity, heredity, teething time, etc. (Reich E. Prevention of caries today / New in dentistry. -2011. - No. 6 (178). - C 6-15 .; Slayton R. Risk assessment of caries development // Maestro of dentistry. - 2007. - No. 25. - C 23-26; Puzikova O.Yu. Prediction of the development of dental caries taking into account integrated indicators and mathematical modeling: Ph.D. in medical sciences - Omsk, 1999. - 183 p .; J. Tsubonchi, et al. A study of dental caries and risk factors among Native American infants / // J Dent Child. - 1995. - Vol. 62. - P. 283-287).

The transition from normal to pathology is usually denoted by prenosological conditions, the recognition of which, called the “prenosological diagnosis”, is the basis for the formation of health groups and the development of primary preventive measures (Vinogradova TF, 1977; Okushko V.R., Kosareva L.I. , 1983; Udovitskaya E.V. et al., 1983). Today, attempts are being made to prognostic prediction of the carious process in children, using objective indicators of oral homeostasis. A special place in order to predict dental pathology in children is occupied by the study of a number of saliva indicators (Suntsov V.G., 1987; Leontyev V.K., Petrovich Yu.A., 1976). An attempt was made to study the immunological status of oral fluid. A search is underway for an immunological marker for dental caries in children with the aim of predicting the disease at the prenosological stage of development (Risik of aktoren ftirdas Nursingottle-yndrome / Ch. Hirsh [et al.] // Oralprophylaxe. - 2000. - Vol. 22. - P. 103-07; Robko FJ Haufigkeit der Nuckelflaschen karies beiVorschulkindern in einer westdeutschen Grobstadt / FJ Robko, M. Buitkamp // Oralprophylaxe. - 2002. - Vol. 24. - P. 59-65; Sellmann HH Kariesrisikotest jetzt auch als DNSSonden // Dental Spigel. 2003. Vol. 1. - P. 44-45; van Palenstein Helderman WH Rise factors of early childhood caries in a Southeast Asian population / WH van Palenstein Helderman, W. Soe, MA van't H about / / J. Dent. Res. - 2006. - Vol. 85, No. 1. - P. 85-88; Gorbunova I. L. Using DNA technology for early detection predisposition to caries / Dentistry - 2006. - No. 4. - P. 18-19; R ost and the development of the child / V.V. Yuriev et al. - 2nd ed. - St. Petersburg: Liter 2003. - 272 p.). Along with the above methods of prenosological diagnosis of dental pathology at different times, the following were proposed: a method for determining the resistance of caries by plaque flora (Kuskova VF, Rebreeva LN, 1971); by titer of lactobacilli of the oral cavity (I.A. Begelman, 1964); according to the buffer capacity of saliva (Sidney, 1963). Mass pre-nosological diagnosis should be one of the most important elements in the system of protecting children's health, as it is aimed not at finding a disease, but at establishing a level of health (Suntsov V.G., Leontyev V.K., Distel V.A., Wagner V. D. Dental prophylaxis in children, Moscow, 2001.344 s.).

In connection with the foregoing, our studies are devoted to the determination of the titer of Streptococcus mutans bacteria in biotopes of the oral cavity in young children.

Currently, rapid methods are used to study the microbial composition of the oral cavity and the integral parameters of saliva, which are quite simple and convenient for children to use directly in the dental office: “Dentobuff Strip”, “Dentocult SM Strip mutans”, “Dentocult LB” (“ Vivadent ”) and“ Saliva-Check Buffer ”,“ Saliva-Check mutans ”(“ GC ”). They help collectively evaluate homeostasis of the oral cavity and predict the development of the carious process in childhood. In a clinic, this allows you to individually prescribe preventive measures and significantly increase the effectiveness of their implementation. Predicting the risk of caries in a child has significant potential in its prevention and treatment, including the organization of dynamic monitoring.

In vitro diagnostic tests for children are used for microbiological testing: Steptococcus mutans - “Dentocult SM Strip mutans” (Vivadent, Liechtenstein); "Saliva-Check Mutans" (company "GC", Japan).

For the Dentocult SM Strip mutans rapid test, the rough surface of the test strip with a rounded end is pressed against the tongue of the child in which saliva is present. Test strips with a square end have a rough surface on which plaque is applied from tooth enamel. Then the strips are placed in selective nutrient saliva in a vial with a previously dissolved disk of the antibiotic bacitracin, which inhibits the growth of bacteria, except for S. mutans. The bottle is placed in an incubator for 48 hours at a temperature of 37 ° C. After the incubation, the presence of bacteria is detected by growth on the rough surface of the test strip of colonies having a color ranging from dark blue to blue. The quantitative composition is characterized by the density of their attachment and is compared with a reference card / scheme.

A known method of sampling material for the study of microorganisms of the oral cavity, the Saliva-Checkmutans test kit allows a semi-quantitative analysis of the concentration of S. mutans in saliva using a highly specific immunochromatographic method. At high concentrations of S. mutans in saliva, bacteria react with gold-labeled colloidal anti-S. mutans, monoclonal antibodies, which are contained in the testing device. The resulting bacteria react with other anti-S. mutans antibodies, which leads to the appearance of a positive red line in window T. Labeled anti - S. mutans / monoclonal antibodies that have not reacted form a control red line in the control window C. Saliva in children is collected in a container up to line A, in children under 3 years old, a special swab on the rod is used for this. In a container with saliva in an upright position, a drop of reagent N21 / alkaline solution is added to dissolve the viscous components of saliva) and mix thoroughly by tapping the container for 10 s. Then 4 drops of N22 reagent / acid solution are added to change the pH of the solution. In this case, the color of the resulting solution changes to light green. The required amount of solution is taken from the container with a pipette and squeezed out into the sample window on the testing device; a wide red strip appears in the control window. After 15 minutes, the results are evaluated. The concentration level is regarded as high -> 5 × 10 ⁵ CFU / ml, if a thin red line appears in the test window “T”. If the line does not appear, then the level of concentration of S. mutans in saliva is low. The test result is considered positive even if a faint red line appears in the "T" window.

There is also known a method of sampling material for the study of microorganisms of the oral cavity (Kuskova V. F., Rebreeva L. I., "Dentistry", No. 4., M., 1971, S. 57-60), in which the material is taken with a thin cotton turundas on root needles or balls of diameter from 2 to 5 mm tightly twisted from cotton wool, previously sterilized in Petri dishes or paper bags by dry air at 165-170 ° C for 1 hour. Moor the turunda or cotton ball immediately before taking a sample with a touch of sterile saline in vitro. Immediately after taking the sample, the turundu, taken with tweezers from the root needle, the cotton ball is lowered into a test tube with a nutrient medium. In this case, the usual bacteriostatic technique of crops over the flame of the burner is observed. To wash out germs in the saliva from those areas where they accumulate in the greatest amount - interdental spaces, the surface of the root of the tongue, gingival pockets, the following two methods can be applied. The first method: the subject is allowed to chew pieces of sterile wax or paraffin / with a volume of about 5 cm ³ /. Sterilization is carried out in an autoclave in large portions in flasks. Before use, paraffin or wax is melted in a water bath, poured into sterile Petri dishes and cut with a sterile instrument. It is enough to choose 5-6 ml of saliva. Immediately before stimulation, the subject rinses his mouth with boiled water.

The second method: the subject pre-rinses his mouth with 50-100 ml of sterile saline to remove food debris and stimulate salivation. After 10-15 minutes, the subject is allowed to rinse his mouth with 8-10 ml of sterile saline and take rinse liquid diluted with saliva in this way. Rinse should be strong and long, up to 3 minutes. The rinse should wash your mouth thoroughly. In both methods, saliva is taken into wide-necked tubes or cones sterilized under double caps, which ensures the sterility of the edges of the glass, which the subject can touch with her lips. The same precaution is needed when sterilizing test tubes with saline rinse. When the sample is taken, the paper cap is replaced with a cotton stopper. Samples of saliva are stored in the refrigerator without freezing, no longer than 4 hours.

A known method of sampling material for microbiological studies of the oral cavity and a device for its implementation, according to patent No. 2101692 Zufarova S.A. and coauthors. The inventive rinse the mouth with sterile saline solution, take samples, cultivate them and identify the microorganisms of the oral cavity in a standard set of environments. Microflora is taken from local areas of the mucous membrane and dentures directly into sterilized metal sleeves with the nutrient medium located there. A device for sampling material for microbiological studies of the oral cavity contains a sterilized container, which is made in the form of a metal sleeve placed in a sleeve holder connected to the head, a handle, the handle being connected to the head shaft with axial rotation, the sleeve holder is pivotally attached to the head. The head is made elastic with the possibility of adjusting the friction in the hinge, and the sleeve has an element of localization of the sample in the form of a nose with sharp edges. The swivel joints of the head with the sleeve holder are made in the form of a semi-ring elastic plate, inside of which the sleeve holder is suspended on two axles, while one of the axes is provided with a thread and an adjusting screw. The method allows to obtain better samples from the local surface of the oral cavity and prostheses and to increase the reliability of microbiological analysis, convenient in operation.

Known methods of collection from the back of the tongue and bacteriological examination of the scraping material of the mucous membranes from the lesion, described in the textbook "Microbiology, Virology and Immunology of the oral cavity" edited by Professor V.N. Tsarev, publishing group GEOTAR-Media, chapter 10. General rules and regulations of microbiological research of various biotopes of the oral cavity.

These methods and the means used for its implementation are accepted as the closest prototype analogues. However, all of the above methods have significant disadvantages. All of them are of little use for the study of cariogenic flora in young children; they do not provide an accurate method for collecting material from the root and back of the tongue, an algorithm for seeding it, choosing nutrient media, bacteriological diagnosis and identification of active microflora as a model system for determining the presence and titer of cariogenic S. mutans using modern culture media. Also a disadvantage of the above prototype analogues is: time costs, lengthy manipulation, high cost of specialized labor. Due to the peculiarities of the psychosomatic status of young children, their restlessness, the collection of material from tooth surfaces is inconvenient. In this regard, it is necessary to choose a biotope and the optimal method, which involves minimal time costs for taking biomaterial, convenient, harmless, and easily accessible in young children.

In the available scientific literature there is no indication of the relationship and interdependence between the dissemination of cariogenic flora of biotopes of the mucous membrane of the back of the tongue and plaque from hard surfaces of teeth and saliva in young children.

We conducted microbiological studies and proposed an improvement in the standard method using a ready-made selective medium for saliva collection not only from the tongue, but also from other biotopes of the oral cavity. At the same time, we divided the standard test strip into 4 parts, this area on its rough surface is enough to reveal the level of streeding of Streptococcus mutans and to conduct a comparative analysis of the growth density of colonies from 4 different actual biotopes of the child's oral cavity.

In the course of our study, it was found that the density of the seeding of cariogenic flora of the lingual surface of the teeth and the mucous membrane of the back of the tongue are statistically significantly identical.

An analysis of the colonization density of various surfaces of the teeth, the interdental space and the back of the tongue by pathogenic streptococci, is shown in Table 1; it showed the predominance of Streptococcus mutans on the lingual surface of the teeth and the mucous membrane of the back of the tongue, with the lowest concentration of microorganisms on the vestibular surface (p <0.001).

The objective of the invention: to propose an indirect method of bacteriological diagnosis for detecting the contamination of Streptococcus mutans plaque in deciduous teeth in children by determining cariogenic microorganisms Streptococcus mutans on the mucous membrane of the back of the tongue using ready-made tests and a selective medium devoid of the above disadvantages.

The technical result consists in the fact that a gentle diagnostic procedure is performed for young children, with the possibility of indirectly detecting streptococcus mutans contamination of the oral cavity in young children by determining the presence, as well as the number of pathogenic cariogenic microorganisms S. mutans, from among the indigenous microflora of the human oral cavity, with easily accessible for sampling biomaterial of the mucous membrane of the back of the tongue, without the use of direct sampling of plaque from hard surfaces of milk teeth and its bacteriological Skog study.

The quality of diagnostics improves, with the possibility of repeated bacteriological monitoring of the condition of the mucous membranes of the back of the tongue as an indirect way to detect cariogenic bacteria Streptococcus mutans biotope of the lingual surface of the teeth in children to monitor the results of the therapy. Development of an individual program to prevent the development of the carious process by means of bacteriological control in the process of prevention and treatment with the possibility of correction of preventive / therapeutic procedures in children. The absence of obstacles to the repeated collection of biological material from the surface of the tongue at the appointment with a dentist or other specialist makes it possible to widely use the proposed method for identifying pathogenic flora in the tongue microbiome as a predictive model for determining Streptococcus mutans in the oral cavity in young children. Early prevention of caries in children plays an important role in maintaining dental health.

The technical result is achieved by the fact that before the start of preventive and therapeutic measures, a microbiological study of young children is carried out, determining the presence and density of bacteria in the mucous membrane of the dorsal surface / back of the tongue by bacteria Streptococcus mutans. The fence is carried out with a sterile tupfer from the entire surface of the mucous membrane of the middle third of the back of the child’s tongue. The degree of colonization by streptococci mutans of plaque on the lingual surface of milk teeth is assessed by the presence and quantitative content of Streptococcus mutans of the microbiome of the mucous membrane of the back of the tongue as a prognostic model using the non-claimed microbiological test "Dentocult SM Strip Mutans" manufactured by Vivadent, Liechtenstein. The test is based on the use of a selective nutrient medium for cariogenic streptococci and a test strip with a square end and a rough surface, based on the natural ability of Streptococcus mutans bacteria to adhere. To conduct a rapid test "Dentocult SM Strip Mutans", the manufacturer recommends applying biological material on the rough rather than smooth surface of the test strip.

The strip with the applied biological material from the mucous membrane of the middle third of the back of the tongue is immersed, according to the manufacturer’s instructions, in a Streptococcus mutans selective nutrient broth with antibiotic bacitracin, to which S. mutans are resistant. The vial is incubated for 48 hours at a temperature of 37 ° C. After the incubation, the presence of bacteria is detected by the growth density of the colonies towering on the rough surface of the test strip in the form of small blue balls with a diameter of 0.1-0.5 mm. Evaluation of the results is carried out according to the attached typical scheme in the instructions for use, reflecting the number / density of Streptococcus mutans colonies, which belong to one of four classes from 0 to 3. The results are recorded in the register of selected strains. The number / density / contamination / titer of S. mutans detected in the microbiome of the mucosa of the back of the tongue statistically significantly corresponds to the titer of Streptococcus mutans in the plaque of the most biotope of the lingual surface of deciduous teeth that they see. Therefore, the obtained data of the tongue microbiome can be used as a prognostic model for determining the dissemination of hard tooth tissues in young children by cariogenic bacteria Streptococcus mutans.

Our research work was carried out for 3 years in 2013-2015. Clinical and instrumental examination of 53 organized children of 3 years of the city of Khabarovsk was carried out. All children who participated in the study did not show any health complaints. Informed consent was previously obtained from parents in accordance with Federal Law No. 323 of February 21, 2011 - ФЗ Article 20. Before starting microbiological diagnostics, all children underwent standard dental examinations and evaluated CPD, hygiene index, and cariogenicity of dental plaque. To calculate the hygienic state of the oral cavity, an index was used according to E. M. Kuzmina, 2000, for young children. The cariogenicity of plaque was determined according to the method of Hardwick J.L., Manley E.V. in modification Nedoseko VB et al. (Nedoseko V.B., Lomiashvili L.M., Poselyanova I.V. Modification of the method for determining cariogenicity of plaque // Dental caries and its complications. - Omsk, 1991, S. 30-31). The results were evaluated by the intensity of plaque staining in points: yellow - 1 point, orange - 2 points, red -3 points.

For microbiological diagnosis, sterile tuffers were used, taking 4 sterile swabs from 4 oral biotopes: namely, from the vestibular and lingual surfaces of the teeth, the interdental space of the milk teeth, and also the mucous membrane of the dorsal surface / back of the tongue, applying the test material to one of the four indicated areas of the test strip.

The standard test strip was divided into 4 parts. This area on a rough surface is enough to apply biological material and to reveal the level of contamination of Streptococcus mutans and to conduct a comparative analysis of the growth density of the colonies immediately in 4 different biotopes of the child's oral cavity with each other. Plaque is taken from an intact area of tooth enamel.

After applying the biomaterial to the test strip, it was immersed, according to the instructions, in a blue-green nutrient broth selective for Streptococcus mutans in a vial with the antibiotic bacitracin disk placed in it 15 minutes before inoculation, which inhibits the growth of all bacteria except S. mutans . The vial was incubated for 48 hours at a temperature of 37 ° C. After incubation, the presence of bacteria was detected by the growth density of colonies, towering on the rough surface of the test strip, in the form of small blue balls with a diameter of 0.1-0.5 mm, while their color varied from dark blue to blue. Colonies dissolved in a selective nutrient broth were not included in the evaluation of the result, which was carried out according to the attached standard scheme in the instructions for use, reflecting the number / density of Streptococcus mutans colonies, which belong to one of four classes: class 0 corresponds to less than 10,000 colonies (CFU <10 ⁴ / ml), 1st grade, less than 100,000 colonies (CFU <10 ⁵ / ml), 2nd grade - from 100,000 to 1,000,000 (CFU10 ⁵ - CFU10 ⁶ / ml), 3rd grade - > 1,000,000 colonies (CFU> 10 ⁶ / ml).

The growth pattern and seed density of Streptococcus mutans in the 4 zones of the test strip are shown in FIG. 1-4.

The studies revealed a slight contamination of the vestibular surfaces of the teeth and a significant contamination of streptococci mutans of the lingual surface of milk teeth and the mucous membrane of the back of the tongue, quantitatively comparable to each other, and to a lesser extent the contamination of the interdental spaces in young children. Seeding data of Streptococcus mutans are presented in Table 1 and in FIG. 5.

The advantages of the proposed method are:

- harmlessness, reduction of time costs, profitability;

- accessibility of the biotope of the back of the tongue in children;

- sparing diagnostic procedure for young children; ease of use;

- the possibility of an indirect diagnosis of dental plaque caries in young children by identifying cariogenic microorganisms from the indigenous microflora of the human oral cavity, Streptococcus mutans, from the mucous membrane of the back of the tongue, without the use of direct collection of soft plaque from hard tooth surfaces and its bacteriological examination;

- improving the quality of diagnosis, with the advent of the possibility of repeated bacteriological monitoring of the contamination of the mucous membranes of the back of the tongue as an indirect / indicative way to identify cariogenic bacteria as a result of a set of preventive measures;

- prevention of dental caries in young children through bacteriological control during treatment / prophylactic measures and the possibility of correction of therapeutic procedures;

- the absence of obstacles to the repeated collection of biological material from the surface of the back of the tongue at the appointment with a dentist or other specialist, makes it possible to widely use the proposed method for detecting cariogenic flora in the root microbiome as a prognostic model for determining the incidence of hard tooth tissues by cariogenic bacteria in young children;

- after completing the course of the complex of treatment and prophylactic measures, in the absence of contraindications, it is possible to conduct double bacteriological monitoring of the condition of the mucous membranes: the surface of the back of the tongue and lingual surface of the teeth;

- the availability of the biotope of the oral cavity; there are no psychogenic reactions, stress in young children when using the proposed method;

- there are no contraindications for the application of the method; the ability to use the method both in combination with other methods, and without them.

Example 1

Child A., 3 years, 2 months, during initial treatment revealed: lack of sanitation - teeth CPD was 6, poor oral hygiene / GI - 0.14 and pronounced cariogenic plaque - 2 points. The child was examined microbiologically, according to the proposed method. The biomaterial was taken from 4 biotopes: plaque from the vestibular and lingual surfaces of the teeth, the interdental space and from the mucous membrane of the back of the tongue. It was found that the contamination of the mucosa of the back of the tongue and lingual surface of the teeth with cariogenic bacteria Streptococcus mutans corresponds to the 3rd class / CFU> 10 ⁶ / ml; No statistically significant differences were found. The seeding of the vestibular surface of the tooth is 1 class / CFU <10 ⁵ / ml, interdental space - 2 class, which corresponds to CFU 10 ⁵ / ml - CFU 10 ⁶ / ml.

Example 2

Child R., 2 years 11 months, tooth CPD is 4. Poor oral hygiene / GI - 0.14 and pronounced cariogenic plaque - 2 points. The child was examined microbiologically, according to the proposed method. The biomaterial was taken from 4 biotopes: plaque from the vestibular and lingual surfaces of the teeth, the interdental space and from the mucous membrane of the back of the tongue. It was found that the contamination of the mucous membrane of the back of the tongue and lingual surface of the teeth with cariogenic bacteria Streptococcus mutans was in the 3rd class, which corresponds to CFU> 10 ⁶ / ml; No statistically significant differences were found. The seeding of the vestibular surface of the tooth is 1 class / CFU <10 ⁵ / ml, the interdental space is 1 class, which corresponds to CFU <10 ⁵ / ml.

Example 3

Child O., 2 years, 9 months, tooth CPD = 4. Poor oral hygiene / GI - 0.14, the value of cariogenic plaque - 1 point. The child was examined microbiologically, according to the proposed method. The biomaterial was taken from 4 biotopes: plaque from the vestibular and lingual surfaces of the teeth, the interdental space and from the mucous membrane of the back of the tongue. It was found that the contamination of the mucous membrane of the back of the tongue and lingual surface of the teeth with cariogenic bacteria Streptococcus mutans in the 3rd class, which corresponds to CFU> 10 ⁶ / ml; No statistically significant differences were found. The seeding of the vestibular tooth surface is grade 2 / CFU <10 ⁵ / ml, the interdental space is grade 3, which corresponds to CFU> 10 ⁶ / ml.

Example 4

Child S., 3 years old, tooth kpu is equal to 7. Poor oral hygiene / GI - 0.14, the value of cariogenic plaque - 2 points. The child was examined microbiologically, according to the proposed method. The biomaterial was taken from 4 biotopes: plaque from the vestibular and lingual surfaces of the teeth, the interdental space and from the mucous membrane of the back of the tongue. It was found that the contamination of the mucous membrane of the back of the tongue and lingual surface of the teeth with cariogenic bacteria Streptococcus mutans was - Grade 3, which corresponds to CFU> 10 ⁶ / ml; No statistically significant differences were found. The seeding of the vestibular tooth surface is grade 3 CFU> 10 ⁶ / ml, the interdental space is grade 3, which corresponds to CFU> 10 ⁶ / ml.

Example 5

Child S., 2 years 4 months, tooth CPD is 0. Oral hygiene / GI - 0.8, the value of cariogenic plaque - 1 point. The child is examined microbiologically according to the proposed method. The biomaterial was taken from 4 biotopes: plaque from the vestibular and lingual surfaces of the teeth, the interdental space and from the mucous membrane of the back of the tongue. It was found that the contamination of the mucous membrane of the back of the tongue and lingual surface of the teeth with cariogenic bacteria Streptococcus mutans was - 1st class, which corresponds to CFU <10 ⁵ / ml; No statistically significant differences were found. The seeding of the vestibular surface of the tooth is class 0, which corresponds to CFU <10 ⁴ / ml, interdental space is class 0, which corresponds to CFU <10 ⁴ / ml.

Claims (1)

A method for determining the uptake of cariesogenic bacteria Streptococcus mutans hard tooth tissues in young children, including the study of the biomaterial of the tongue microbiome as a prognostic model before the start of preventive and therapeutic measures, characterized in that the biomaterial is taken from the entire surface of the mucous membrane of the middle third of the back of the tongue with a sterile tuffer, then the biomaterial is applied to standard Dentocult SM Strip Mutans test strips with a square end and a rough surface, placed in a village tive medium for cariogenic streptococci after incubation assess the degree of colonization by streptococci Microbiome mucosal language backrest assigning the number to the classes from 0 to 3, respectively, of CFU <10 ⁴ / ml to CFU 10> ⁶ / ml after referring to the class of contamination S. mutans microbiomes of the tongue, determine if they are seeded by the biotope of the lingual surface of the deciduous teeth, given that the number / density / seeding / titer of S. mutans in the microbiome of the tongue statistically significantly corresponds to the titer of Streptococcus mutans in plaque ychnoy surface deciduous teeth.

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