Microbiocenosis of the oral cavity of children: clinical and microbiological characteristics and correction with probiotics based on salivary streptococci

Introduction

Microbiocenosis of the oral cavity (oral microbiocenosis) is a complicated multicomponent self-regulating system including microscopic representatives of pro-and eukaryotic microbes as well as human viruses and bacteria (bacteriophages). According to the latest data the bacteria (prokaryotes), the number of which is more than 800 species, make up the largest and the most significant part of it from the clinical point of view [1].

The structural and functional arrangement of the oral microbiocenosis is based on the metabolic links occurring in the microbial community that is called “biofilm”. It is facilitated by extracellular digestion that is distinctive for single-cell living things. In other words, the initial splitting of nutrients goes in the microenvironment at the account of exoenzymes produced by various microbes. So, the bacteria get an opportunity of using the split products made by the enzymes of other symbionts of this ecological niche [2, 3].

The biofilm in the oral cavity results from the consecutive process of the initial colonization of residential microaerophilic streptococci (Streptococcus sanguinis, S. oralis, S. mitis, S. gordonii, S. cristatus), veillonelli (Veillonella parvula, V. atypica, V. dispar) and actinomycetes (Actinomyces naeslundii, A. odontolyticus, A. israelii, etc.) attaching the surface of the tooth and the oral mucosa due to various adhesive factors. The mixed, mainly coccal population, is formed. Interim and later colonizing microbes colonize firstly due to the co-aggregation of the bacteria and the initial colonizers and finally due to the attachment of later colonizers to the initial and interim ones [4—7].

In spite of the stability and obvious capability of oral microbiocenosis to self-regulate this process is susceptible to different negative exogenous and endogenous effects [6—8]. It faces significant changes in cases of teeth eruption, primary teeth replacement by permanent ones, hormonal changes in adolescence that can lead to multiple caries, gingivitis and periodontitis [8—12]. These risk periods require more attention of the dentist and some correcting measures to support the hygiene status of the dentitions and microbial balance using pre- and probiotics. The excreted probiotic strain M18 Streptococcus salivarius offers the potential to confer oral health benefits as it produces salivarcins — antibacterial substances of local action that are capable to restrict the growth of infectious agents in the oral cavity: Streptococcus spp., Porphyromonas spp., Actinomyces spp., Aggregatibacter spp. The strain M18 also produces enzymes: dextranase and urease reducing the amount of dental plaque and neutralizing the acidity in the oral cavity [13, 14]. Today probiotics cause no side effects so bacterium therapy with probiotic strains having inhibiting effects on pathogenic microorganisms of the oral cavity is a promising concept especially for the childhood.

The objective of our study is to assess the stability of the oral microbiocenosis in preschool children according to microbiological parameters and analyze possible correction of changes with probiotics based on the strains of salivary streptococci.

Materials and methods

10 children (7 girls and 3 boys) suffering from multiple caries were under study. All of them were included in the treatment programme — a three-month course of orally disintegrating probiotic DentoBlis (Medico Domus, Serbia). All the children were carried out complete sanitation of the oral cavity.

The standard clinical examination was made: to define Fedorov and Volodkina Index (OHI), to determine the severity of periodontal diseases using the PMA index and the caries intensity of primary teeth using the DMFT index.

In order to assess the oral microbiome, the cultural (bacteriological) method was used. Some samples of microbiome film were taken with a standard swab from the enamel surface in the area of the gingival attachment. The swab was put in the liquid Stuart transport medium to get the laboratory where quantitative cultures in the dense culture media (Himedia, India) were performed: 5% bloody agar with hemin and menadione, chromogenic mitis-salivarius agar and a different diagnostic chromogenic medium for the excretion of the yeasts of the genus Candida. The cultivation and identification of the excreted strains was made through a standard method using anaerostat. The quantity calculation of the colonies was carried out using an automatic method with Scan-500 (Interscience, France).

The methods of variational statistics and the Mann–Whitney criterion (p<0.05) were applied for statistical analysis.

Results and discussion

The examined children showed the caries intensity of 7.1 and Fedorov and Volodkina index (OHI) — 4.08 that was very poor. The PMA index was 61.52% signifying moderate severe gingivitis. The analysis of the indices of the microbial load was made starting from the diagnostically significant figures >100 CFM. Other figures were excluded from the study as statistically insignificant. The data made it possible to assess 12 taxonomic groups of the microbiota of the dental plaque samples (biofilm) that were the most widespread both in terms of prevalence and quantitative representation (see Table).

The most common both in terms of prevalence and quantitative representation was the association of microaerophilic streptococci. S. sanguis and S. mutans were the main acid-producing species and all the samples contained them, their average number was 7.01±0.13 and 6.40±0.11 lg CFM/ml. The number of acid-producers was higher than the established physiological norm — 5.0—6.0 lg CFM/ml. Other important acid-producing components of oral microbiocenosis are actinomycetes and they were also found out in every second child (50%) — 6.03±0.02 lg CFM/ml in average that was higher than the established physiological norm too. At the same time the main stabilizing species producing no acid — S. salivarius — was excreted only in 70% of the examined children — 5.67±0.30 lg CFM/ml corresponding to the physiological norm.

Such stabilizing species antagonizing the cariogenic microbiota due to the utilization of metabolic acid products (Corynebacterium spp. and Veillonella spp.) were found out only in 20 and 60% correspondingly.

The findings testify to a rather high acid-producing potential of the microbiota in the examined children and reflect oral microbiocenosis destabilization causing an increase of cariogenic activity.

The next group of the microbiota of the examined children consisted of periodontal pathogenic species (gram-negative anaerobic bacteria) as well as representatives of the aggressive microbiota (staphylococci and the yeasts of the genus Candida). The findings showed the excretion frequency of periodontal pathogenic species of the so-called “red complex” (P. gingivalis and P. intermedia) in 20% that testified to a further high risk of chronic periodontitis. Moreover, these species were found out in rather large amounts — 5.51±0.12 and 6.00±24 lg CFM/ml correspondingly (taking into account the fact that no such species are identified in healthy periodontal tissues).

70% of the examined children had the aggressive species S.aureus and almost a half (40%) — S. epidermidis in a rather large amount that might signalize some inflammatory process. 1/3 of patients showed a diagnostically significant number of Candida spp. (4.87±0.14 lg CFM/ml) that correlates with the data in the literature about the role of the yeasts in the pathological development of the oral mucosa in children [17].

After the treatment with the probiotic based on S. Salivarius M18 the OHI in children decreased by 2.2 times (the average figure for the group was 1.82 that corresponds to the satisfactory hygiene), the periodontal PMA index — by 2.4 times (the average figure — 25.2%). The quantity indicator of S.sanguis came to the norm while that of S. mutans reduced both in terms of prevalence (till 70%) and in average microbial load (4.92±0.17 lg CFM/ml). It is also important that the frequency of S. salivarius excretion increased till 90% while quantity parameters were saving steadily corresponding to the physiological norm that is explainable by probiotic strain taking. Nevertheless, the other acid-producing group — actinomycetes — was cut down significantly both in terms of prevalence (till 20%) and microbial load (5.00±0.21 lg CFM/ml). The strains antagonizing acid-producers — Corynebacterium spp. and Veillonella spp. — were at the same level and in the same amount.

The essential point to assess the effectiveness of the therapy is a synergy process of the main symbionts. It is fundamental in modern concepts of probiotic strain use for preventive purposes and treatment [9, 11, 12]. It is proved that most of Veillonella spp. excreted out of the oral mucosa of the cheeks and the tongue (42 and 55) has co-aggregative characteristics. Out of 24 Veillonella spp. excreted from the sub-gingival plaque samples 20 species looked like the following: V. parvula co-aggregating with A. viscosus, A. naeslundii, A. israelii, S. sanguis, F. nucleatum and other bacteria presenting in the oral microbial community. All the species of Veillonella spp. co-aggregated with S. salivarius [18]. This information was used in our previous study to make a test complex product based on the strains of S. salivarius K-12 and V. parvula that showed high antagonistic activity towards staphylococcus and enterococcus association in the experiment on rats [19].

The previous studies of W. Distler et al. revealed some symbiosis between Veillonella spp. and streptococci in the plaque. Moreover, producing lactate the streptococci act as a nutritive resource for veillonellas. Co-habiting with Veillonella spp. induced expressions into S. gordonii α-amylase that let streptococci disintegrate starch into oligosaccharides and, therefore, metabolize them into lactate that further may be used by Veillonella spp. On one hand it is clear that Veillonella spp. can serve as a basis to attach such virulent periodontal pathogenic species as P. gingivalis and F. nucleatum/periodonticum. On the other one, F. nucleatum in principle co-aggregates with all initial and later colonizers. Among the latter there are periodontal pathological species providing actively inflammatory processes including a teeth eruption period (A. actinomycetemcomitans, P. gigivalis, Treponema spp., Eubacterium spp.) [9].

The study of the effectiveness of probiotic strain S. salivarius M18 showed a statistically reliable reduction of quantity indicator of prevalence of P. gingivalis and P. intermedia although there was no full eradication of periodontal pathogens. It also illustrated a decrease in the excretion frequency of the aggressive strain of staphylococci — S. aureus (from 70% to 10%) and the yeasts of the genus Candida (from 30% to 10%). The data proved the findings of randomized controlled trials to test clinically products based on the probiotic strain S. Salivarius M18 [21, 22].

It is notable that S. salivarius M18 can aid neither in pH reduction nor in dental plaque formation in the oral cavity due to its natural enzyme structure. Such enzymes are urease and dextranase in particular: urease splits urea restoring pH to the physiological norm and dextrose splits dextran that is a basic component of the plaque and a reserved nutrient for S. mutans. So the metabolic activity of S. salivarius contributes to cariogenic microbial biofilm destruction and facilitates further dental plaque formation [21, 22].

At the same time modern molecular genetic methods proved safety of this probiotic strain as its genome was fully sequenced and no plasmids, transposons or any other generic determinants coding the synthesis of pathogenic factors were found out in it. There is evidence that S. salivarius M18 has antimicrobial activity to combat S. mutans and other infection pathogens in the oral cavity including the periodontal pathogenic species P. gingivalis, A. acinomycetans and F. nucleatum that is proved by clinical laboratory data presented in the study. This activity of S. salivarius M18 might be explained by producing specific antimicrobial agents — Salivarcins M, A2, 9 and MPS [23, 24]. Thus, the results of the study testify to negative trends of the oral health of the examined children and these trends are linked with the changes in the content of the oral microbiocenosis and may be of dual character. The first trend advances towards the progress of acid-producing microbiota (S. mutans, S. sanguis, Actinomyces spp.) and the other one — towards periodontal and another aggressive microbiota (P. gingivalis, P. intermedia. S. aureus, Candida spp.).

Conclusion

The obtained results of the microbiological study correlate to the clinical picture of the examined children with primary teeth caries and inflammatory periodontal diseases. DentoBlis caused a significant (in 2.2 times) improvement of the OHI and a reduction of gingivitis prevalence (PMA index) by 36%.

The probiotic product based in the strains of S. salivarius M18 is promising to correct and stabilize oral microbiocenosis as the evidence-based increase in colonization frequency of salivary streptococci is accompanied with a cut of acid-producing and periodontal pathogenic potential of microbiocenosis as well as a rise of excretion of stabilizing microbiota representatives.