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DOI: http://dx.doi.org/10.20534/ESR-17-3.4-41-44
Kazakova Larisa Nikolaevna, Saratov State Medical University n. a. V. I. Razumovsky, epartment of Children Stomatology and Orthodontics,
PhD in Medical Science, E-mail: [email protected] Zholdasova Nurgul Zhanabayevna, Candidate of Medical Sciences, Department of Surgery and Children Stomatology, West Kazakhstan Marat Ospanov State Medical University,
E-mail: [email protected] Suetenkov Dmitry Evgenievich, Saratov State Medical University n. a. V. I. Razumovsky, Department of Children Stomatology and Orthodontics, Assistant Professor, PhD in Medical Science, E-mail: [email protected]. Kaikan Azamat Islambekuly, Master of Medical Sciences, Department of Surgery and Children Stomatology, West Kazakhstan Marat Ospanov state medical university, assistant,
E-mail: [email protected] Mahonova Ekaterina Vladimirovna, Saratov State Medical University n. a. V. I. Razumovsky, Department of Children's Stomatology and Orthodontics, Assistant,
E-mail: [email protected].
Microflora of biotopes as a leading etiological factor at oral diseases in children
Abstract: Being a source of foreign genetic information, microbes and viruses regardless of the exact place of the invasion during their life damage tissues' integrity and as a result provoke the development of disease of various severity. Oral cavity stands on the second place by the number of microbes with different level of virulence. Dysbiotic abnormalities cause biofilms formation in various oral biotopes. The associated links within biofilms facilitate the increase of microflora virulence. Poor oral hygiene stimulates microbes' growth and reproduction. Oral cavity is not homogeneous by the number of microbes' settlements. Most of microbes concentrate in areas with special anatomical features. Stomatitis, dental caries, parodontitis and periodontitis are the most frequent clinical manifestations of high active microflora. Monitoring of oral biocenoses allows to perform the analysis of their variety and structural adjustments, which is vital for optimal treatment and prevention of oral diseases. Keywords: Microbes, Anaerobe, dental caries, oral hygiene, children, stomatitis.
The most frequently spread somatic diseases are stomatological ones. Every child knows what stomatitis or dental caries is. The main factor, which causes stomatitis in children is the oral bacterial flora [1]. Normally the inflammatory response is prevented by specific and nonspecific factors of antimicrobial resistance of oral mucous membrane, as well as biochemical and immunological features of saliva. The forming of oral micro-biocenosis is a multilevel process, closely linked with the regularities of dento-facial system development. Transient microbes constantly getting into the children's mouth by dirty hands, toys and kisses of relatives damage micro-ecological and adaptive microbes' interrelations with both environment and each other.
Clinical picture of inflammation in the particular patient depends on the factor, causing it as well as on the individual conditions of the patient, complementing this pathological process [2].
Understanding of the oral pathological processes in children requires knowledge of topographic, anatomic, histological and his-tochemical processes, often sharply changing depending on age.
There are distinguished 3 periods of age, which significantly differ by constitution and characterize the dynamic of main mucous structures development: the infants - a period of age from 10 days to 1 year, early childhood - the children from 1 to 3 years old and childhood - primary (4-7 years old children) and postprimary (812 years old children). With age the factors of local mucous protec-
tion improve [3; 4]. And, in spite of quality changes of oral mucous membrane structures during childhood, characterized by principally new ways of defence building, stomatitis is remains a very frequent disease among the children.
Catarrhal, acute and aphthous stomatitis are very similar by their symptoms among the children, therefore inadequate complex treatment of a trivial form of stomatitis often provokes its shifting into aphthous stomatitis [4; 5; 6]. Up to now, the etiology of stomatitis, specifically which factors are dominant and which propose the development of the disease remains open to discussion.
The identification of the exact etiology of stomatitis in each particular case defines the system of medications and preventive measures as well as the prognosis of the disease recurrence.
The aim of investigation is to enhance the effectiveness of stomatitis diagnostics among the children and specify biocenotic interrelations of staphylococci and streptococci, colonizing biotopes of cheek, interdental gingiva and sublingual area.
Materials and methods. The study included the analysis of 60 kids from 7 to 10 years old from the first and second health groups with negative allergic anamnesis. Parents of children, who took part in the analysis, gave their informed agreement. At the moment of investigation 50 kids had no complaints typical for oral inflammatory diseases. 10 kids had from 2 to 5 elements of inflammation of various oral locations.
The children were divides into 3 groups by the frequency of stomatitis occurrence. The first group included healthy kids with no complaints specific for stomatitis in anamnesis in any period of oral system development (20 patients). The second group included 20 kids having a single visit to the doctor related to stomatitis in anamnesis.
20 kids visiting stomatologist once a year with complaints on painful aphthaes in the mouth formed the third group. Investigation included various methods of analysis such as questioning, oral examination, probing, percussion, hygiene state identification and microbiological analysis of oral biotopes.
During oral examination, probing and percussion there were identified n oral cavity and tooth lines conditions, carious and noncarious changes of hard tooth tissues. The oral hygienic state was estimated by Green-Vermillion (OHI-S) method. Dentobacterial plaque was colored by «Dinal» tablets due to their simple use and no emotional impact on children. Dentobacterial plaque was esti-
mated on teeth, belonging to different groups, on surfaces, the most effected by dental deposits. Numeric representation of diagnostic criteria in this method of analysis [7] characterized the area of dental deposit. The intensity of caries at this stage of mixed dentition was estimated by KP and KPU analysis [8].
The bacteriological analysis was conducted according to general rules of clinical microbiology with definition of aerobic and facultative anaerobic microbes and quantative estimates of the results (primary seeding was done from 10-1-10-5 dilutions of analyzed tissues (material)), required to extract conditionally-pathogenic bacteria [9]. Clear cultures of facultative anaerobic bacteria were grown using five-percent blood agar in exsiccators. The extracted cultures were identified after the number of isolated colonies on solid mediums counting. For bacteria identification a complex of morphological, cultural and biochemical patterns according to Berdgi classification (1980) was used. Biochemical identification of clear streptococcus, enterococcus and staphylococcus cultures was fulfilled by Lachema test systems.
Density ofmicrobes' population was expressed in colony-forming units (CFU). The material for bacteriological analysis was taken from surfaces ofbuccal mucosa, interdental gingival mucosa within the teeth used for hygiene index analysis, oral floor mucosa and aphthaes.
Based on the analysis of caries intensity patients in groups were divided as following: the first group was characterized by low active caries and good oral hygiene, satisfactory structure of hard tooth tissues, correct forms and sizes of the erupting permanent teeth, oral mucosa of standard rose-pink color without any pathological changes. In the second group 8 patients from 20 had medium intensive caries, 4 had high intensive caries and 8 had low intensive caries, oral hygiene in the group was mainly satisfactory. No changes of hard tooth tissue structure, no pathological changes of oral mucosa were revealed within the investigation. The third group included 10 patients having oral aphthaes at the moment of the investigation. Aphthaes were located mostly on buccal mucosa within the necks of teeth, covered by soft dental deposits, and were covered by fibrinous pellicle, tender to palpation. Average value of OHI-S index in this group was equal to 0,5, which corresponded to good oral hygiene level, 2 patients had OHI-S index equal to 1,2. 12 patients had caries of high intensity and 8 patients had caries of medium intensity.
1st group (20 patients) 2nd group (20 patients) 3rd group (20 patients)
Microobes Average, m Occurrence Average, m Occurrence CpegHee, m Occurrence
(CFU) rate (0 < p < 1) (CFU) rate (0 < p < 1) (CFU) rate (0 < p < 1)
S. salivarius 9,57 0,7* 13,57 0,7 16,33 0,9
S. sanguis 10,55 0,9 12,25 0,8 15,7 1,0
S. mitis 9 0,7 16,14 0,7 12,0 0,9
S. mutans 12,33 0,6 13,5 0,8 19,0 1,0
S. viridans 0 0 0 0 4,75 0,4
S. aureus 3 0,1 4 0,1 4,28 0,7
S.haemolyticus 0 0 0 0,1 3,2 0,5
Lactobacter 7,66 0,3 17,13 0,8 16,75 0,8
Enterococcus 9,0 0,5 14,71 0,7 8,7 1
Candida spp. 3 0,1 4 0,5 5,75 0,8
*The occurrence rate characterizes the number of patients in a group which have a particular microbe (0,7 corresponds to 70% of patients, namely 14 from 20 analyzed patients of the 1st group with S. Salivarius revealed).
The results of investigation demonstrated low caries intensity the first group, as well as chronicity of the existing pathological sites and good oral hygiene in 100 per cent cases among the patients of on hard tissues. Patients of the second group had caries of low and
Table 1. - Average values of identified microbial CFU and corresponding frequency of occurrence in gingival margin among the patients of the 1st, 2nd and 3rd groups at 10-4 dilution
medium intensity in 80% of cases and caries of high intensity in 20% of cases. Hygienic index was low in 100% cases. Among the patients of the third group poor hygiene was shown in 20% cases, 80% of patients demonstrated satisfactory hygiene level. However, together with good oral hygiene 40% of children in this group had caries of medium intensity and 60% ofpatients - caries ofhigh intensity. Pathological processes on hard teeth tissues revealed acute development in 90% cases.
The conducted bacteriological analysis revealed a multiple variety and high density of CPU in all analyzed biotopes at primary inoculation. Streptococci appeared to be the most representative microbes.
High CFU density of streptococci remained up to 10-4 dilution of biomaterial, taken from interdental gingival mucosa and gingival margin in all 3 groups of patients (table 1, pic.1). Among streptococ-
ci there were identified Streptococcus mutans, S. sanguis, S. salivarius,. S. mitis and S.viridans with index of dominance equal to 100%.
At 10-4 dilution S. salivarius and S. Mitis were identified in 70% cases among the patients of the 1st and the 2nd groups, in the 3rd group S. salivarius and S. mitis were detected in 90% cases. S. Sanguis and S. Mutans were found in all patients of the 3rd group and 80% patients of the 2nd group at 10-4 dilution. Data analysis based on parametri-cal criteria of Dannet with the 1st group used as control revealed a significant difference of the 1st and 3rd groups by S. salivarius (test significance level a = 0,05, number of degrees of freedom v = 29, number of compared groups l = 3), S. Sanguis (a = 0,05, v = 35, l = 3) and S. mutans (a = 0,05, v = 29, l = 3) indexes. The revealed difference was also proved by nonparametric criteria Kruskall-Wallis and Dann.
Picture 1. Average values of identified microbial CFUs in analyzed biotopes of gingival area (10-4 dilution)
Analysis of biomass in first inoculation demonstrated high density of staphylococcus. Typing allowed to extract Staphylococcus aureus and S.haemolyticus. The highest frequency of occurrence of these microbes appeared in the 3rd group of patients (pic.2a) at all dilutions. 95% confidence intervals for frequency estimates in the 1st and 3rd groups at 10-4 dilution built with the use of binomial distribution with regard to small samples allowed to reveal the significant differences of groups by given microbes and consider these indexes as essential for pathological process prognosis. High CFU density of lactic bacteria and enterococcus in the 3rd group remained up to 10-5 concentration with average lactic bacteria CFU density within gingival margin equal to 5,25 and enterococcus CFU density equal to 7,5.
Frequency index of S.viridans, extracted from aphthaes surfaces, made 100%. This alpha-hemolytic streptococcus was distinguished in 40% patients of the 3rd group within gingival margin at 10-4 dilution (average density equal to 4,75 CFU). It also appeared as 5 CFU among 20% patients at 10-5 dilution. Aerobic microflora of this biotope in analyzed groups appeared to be dynamic and changeable.
The comparative study of bacteria population in biotopes ofbuc-cal, sublingual and gingival areas revealed the highest bacterial number in gingival margin. Gingival biotopes of conditionally healthy patients in all three groups significantly differed by both quantitative and qualitative indexes at maximum dilution (pic. 2a, 2b). The highest CPU density of identified streptococci, staphylococci, entero-cocci and lactic bacteria appeared in the 3rd group. Microbial density was lower in buccal mucosa and the lowest in sublingual area.
High number of residential bacteria at buccal mucous areas remaining at 10-4 and 10-5 dilutions contributes to local immune depression and, therefore, creates favorable conditions for transient microbial contamination, which increase biotope virulence.
The study revealed modifications and adjustments of aphthaes micro-biocenosis, characterized by both the decrease of dominance and environmental significance of the leading symbiotes and the increase of transient microflora frequency.
Transient microbes, been inoculated at stomatitis, have distinctive pathogenicity, which is clinically expressed by pathological formations on buccal mucous membrane. S. Haemolyticus in aphtha's biotope saves dominance up to maximum dilution.
Different degree of various biotopes' contamination in the particular patient is linked with anatomic features of these surfaces and immunological background. Constant sublingual and submaxillary salivatory secretions support high concentration of immune protective factors in sublingual area, which allows controlling the bacteria and preventing possible inflammation in this area. However, the diagnosed dysbiotic disorders in biotopes ofbuccal and interdental gingival areas prove the failure of local oral immunity in general.
In summary, the type of staphylococcus bacteria-carrying and S.viridans in oral diseases becomes a way of microbiological monitoring and detection of high risk groups at a particular oral pathology in compliance with identification of the disease structure, as well as clinical and microbiological factors.
The analyses of ecosystem anaerobic structure demonstrated microbes critical for microflora of microbiocenosis were strepto-different contributions of microbes to oral biocenoses and allowed cocci with different type ratios in analyzed biotopes. to define dysbiotic changes in biotopes. It was proved that dominant
Picture 2 a. Frequency of occurrence (%) of aerobes and facultative anaerobes in analyzed biotopes at 10-5 dilution among the patients of the 3rd group
I Group3, 10-5:Buccal mucosa ■ rp3, 10-5:Gingival margim ■ rp3, 10-5:Sublingual area
Picture 2 b. The bacterial number (CFU) of analyzed biotopes at 10-5 dilution among the patients of the 3rd group
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